Patent application title: MSMB-gene based diagnosis, staging and prognosis of prostate cancer
Inventors:
Lijs Beke (Heverlee, BE)
Monique Beullens (Leuven, BE)
Mathieu Bollen (Haasrode, BE)
Kyrylo Litovkin (Leuven, BE)
Mieke Nuytten (Holsbeek, BE)
Aleyde Van Eynde (Pellenberg, BE)
Assignees:
KATHOLIEKE UNIVERSITEIT LEUVEN, K.U. LEUVEN R&D
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Class name: Chemistry: molecular biology and microbiology measuring or testing process involving enzymes or micro-organisms; composition or test strip therefore; processes of forming such composition or test strip involving nucleic acid
Publication date: 2009-08-13
Patent application number: 20090203010
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Patent application title: MSMB-gene based diagnosis, staging and prognosis of prostate cancer
Inventors:
Lijs Beke
Monique Beullens
Mathieu Bollen
Kyrylo Litovkin
Mieke Nuytten
Aleyde van Eynde
Agents:
TRASKBRITT, P.C.
Assignees:
Katholieke Universiteit Leuven, K.U. Leuven R&D
Origin: SALT LAKE CITY, UT US
IPC8 Class: AC12Q168FI
USPC Class:
435 6
Abstract:
This invention relates generally to a method of diagnosis for
distinguishing between a benign prostate hyperplasia and a prostate
cancer and between a hormone-sensitive and a hormone-refractory prostate
cancer condition and specifically to identification of a hypermethylated
(on CpG and non-CpG dinucleotides) CpG island in the
beta-microseminoprotein (MSMB) regulatory regions surrounding the
transcriptional start site of the MSMB gene as a diagnostic indicator of
prostate cancer (PrCa) and for distinguishing androgen-refractory from
androgen-sensitive prostate cancer.Claims:
1. A method of identifying a prostate cell proliferative disorder in a
human male subject, the method comprising:providing a sample comprising
prostatic tissue, prostatic cells, fluid of the prostate from a human
patient susceptible to a prostate cancer, andanalyzing the sample for the
level DNA methylation of the regulatory region surrounding the
transcription start site (TSS) of a beta-microseminoprotein gene (MSMB
gene), wherein hypermethylation in this region indicates the presence of
prostate cancer cells or is indicative of prostate cancer.
2. The method according to claim 1, wherein the region comprises one or more CpG islands and extends from about 3.0 kb upstream to about 2.2 kb downstream from the TSS of the MSMB gene.
3. The method according to claim 1, wherein the region extends from -3128 bp to -2817 bp upstream from the transcription start site of the MSMB gene.
4. The method according to claim 1, wherein the region extends from -3533 bp to -2734 bp upstream from the transcription start site of the MSMB gene.
5. The method according to claim 1, wherein the region extends from -452 bp upstream to +150 bp downstream from the transcription start site of the MSMB gene.
6. The method according to claim 1, wherein the region extends from 2180 bp to 2390 bp downstream from the transcription start site of the MSMB gene.
7. The method according to claim 1, wherein the region between -13877 and -13583 or between -10528 and -10254 or between -3920 to -3673 or between -3471 and -3141 or -3128 and -2817 base pairs upstream from the transcription start site of the MSMB gene is analyzed for hypermethylation.
8. The method according to claim 1, wherein the region between 1671 and 1996 or between 2180 and 2390 between 5236 and 5616 or between 7670 and 8030 or between 11432 and 11754 or base pairs downstream from the transcription start site of the MSMB gene is analysed for hypermethylation.
9. The method according to claim 1, wherein the presence of hypermethylation of CpG and non-CpG dinucleotides in this regulatory region surrounding the transcription start site indicates the presence of prostate cancer cells or is indicative of prostate cancer.
10. The method according to claim 1, wherein the method comprises: analyzing the level DNA methylation of the regulatory region surrounding the TSS of a beta-MSMB gene in a biological sample isolated from the subject; whereby non-CpG methylation or non-CpG hypermethylation in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication of prostate cancer or for prostate cancer cells' presence.
11. The method according to claim 1, wherein the method comprises: analyzing the level DNA methylation of the regulatory region surrounding the TSS of a beta-MSMB gene in a biological sample isolated from the subject, and further comprises comparing the DNA methylation with the DNA methylation in a control sample and/or a benign prostate hyperplasia sample; whereby increased non-CpG methylation in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer or for the presence of prostate cancer cells.
12. The method according to claim 1, wherein the method comprises: analyzing the level DNA methylation of the regulatory region surrounding the TSS of a beta-MSMB gene in a biological sample isolated from the subject, and further comprises comparing the DNA methylation with the DNA methylation in a control sample; whereby increased CpG methylation in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer or for the presence of prostate cancer cells.
13. The method according to claim 1, wherein the method comprises: analyzing the level DNA methylation of the regulatory region surrounding the TSS of a beta-MSMB gene in a biological sample isolated from the subject, and further comprises comparing the DNA methylation with the DNA methylation in a sample of androgen sensitive prostate cancer; whereby increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to the androgen sensitive prostate cancer is an indication of an hormone refractory prostate cancer, androgen-independent prostate cancer (AIPC) or androgen-independent metastatic prostate cancer.
14. The method according to claim 1, wherein the method comprises: analyzing the level DNA methylation of regulatory region surrounding the TSS of a beta-MSMB gene in a biological sample isolated from the subject, and further comprises comparing the DNA methylation with the DNA methylation in a control sample or a benign prostate hyperplasia sample; whereby increased non-CpG methylation in the regulatory regions surrounding the transcriptional start site of the MSMB gene relative to a control sample is an indication for prostate cancer and comparing the DNA methylation with the DNA methylation in a androgen sensitive prostate cancer sample; and whereby an increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to a control sample is an indication of an hormone refractory prostate cancer, androgen-independent prostate cancer (AIPC) or androgen-independent metastatic prostate cancer.
15. The method according to claim 1, wherein the method comprises:analyzing histone acetylation or deacetylation of the MSMB gene in the sample.
16. The method according to claim 14, wherein the method comprises:analyzing histone acetylation or deacetylation of the MSMB gene in the sample.
17. The method according to claim 1, wherein an androgen-independent metastatic prostate cancer in a prostate cell or prostate tissue is diagnosed.
18. The method according to claim 14, wherein an androgen-independent metastatic prostate cancer in a prostate cell or prostate tissue is diagnosed.
19. The method according to claim 1, further comprising:carrying out a prostate cancer grading or prostate cancer staging.
20. The method according to claim 14, further comprising:carrying out a prostate cancer grading or prostate cancer staging.
21. The method according to claim 1, further comprising:deciding on a treatment or medicament of the prostate disease state.
22. The method according to claim 14, further comprising:deciding on a treatment or medicament of the prostate disease state.
23. The method according to claim 1, wherein hypermethylation is determined using PCR or other amplification technique.
24. The method according to claim 1, wherein hypermethylation is determined by bisulfite genomic sequencing PCR analysis.
25. The method according to claim 1, wherein hypermethylation is determined by Methylation-Specific PCR analysis or other amplification technique.
26. The method according to claim 1, wherein hypermethylation is determined by a diagnostic array, the array comprising primers for assessing the presence of hypermethylation in a regulatory region surrounding the TSS of the MSMB gene.
27. The method according to claim 1 which utilizes at least one primer of the group consisting of methylated specific primers (SEQ ID NOs: 13, 14, 17, 19, 20, 21, 22, 26, 28, 29, 38, 39, 42, 43, 50, 51, 57, 58, 61, and 62) and of the group consisting of unmethylated specific primers (SEQ ID NO's 15, 16, 18, 23, 24, 25, 27, 30, 31, 40, 41, 44, 45, 52, 53, 59, 60, 63 and 64).
28. The method according to claim 1 which utilizes at least one primer of the group consisting of bisulfite sequencing primers (SEQ ID NO: 9, 10, 11, 12, 32, 33, 34, 35, 36, 37, 46, 47, 48, 49, 54, 55, 56, 65, 66, 67, 68, 69 and 70).
29. The method according to claim 1, wherein distinguishing between methylated and non-methylated CpG dinucleotide sequences within the target sequence comprises utilizing at least one primer in each case a contiguous sequence at least 16 nucleotides in length that is complementary to, or hybridizes under stringent conditions to a bisulfite-converted sequence (SEQ ID NO:6 and 8) derived from a sequence selected from the SEQ ID NO:5 and 7.
30. The method according to claim 1 wherein PCR analysis is performed on polynucleotide materials of cells derived from prostatic tissue.
31. The method according to claim 1 wherein PCR analysis is performed on polynucleotide materials of the cells derived from seminal fluid or from ejaculate.
32. The method according to claim 1 wherein PCR analysis is performed on polynucleotide materials of the cells derived from body fluids selected from the group consisting of blood, urine, ejaculates, prostate secretions, and combinations thereof.
33. The method according to claim 1 wherein PCR analysis is performed on polynucleotide materials of the cells derived from prostate tissue from histological slides or biopsies or paraffin-embedded tissue.
34. The method according to claim 1 wherein PCR analysis is performed on polynucleotide materials of the cells of prostatic tissue from biopsy or from surgical resection.
35. The method according to claim 1, comprising: obtaining a biological sample from the subject; determining the methylation state of CpG island upstream and/or downstream of the transcriptional start site of the MSMB gene in the subject's sample; and identifying hypermethylation of one or more CpG islands of the regulatory region surrounding the transcription start site of the MSMB gene, wherein detection of hypermethylation is indicative of a predisposition to, or the incidence of prostate cancer.
36. The method according to claim 1, wherein differential methylation is observed when compared to the methylation status of the regulatory region surrounding the transcription start site of the MSMB gene from a androgen-sensitive prostate cancer cell or a normal cell, which differential methylation is hypermethylation and is indicative for androgen-refractory prostate cancer.
37. The method according to claim 1, wherein detection of hypermethylation is indicative for the grade and/or stage of the prostate proliferative disorder.
38. The method according to claim 1, wherein detection of hypermethylation is indicative for decided about a treatment with a DNA methylation inhibitor.
39. The method according to claim 1, wherein detection of hypermethylation is indicative for decided about the initiation or continuation of treating with a compound in an effective amount to reduce male hormones.
40. The method according to claim 1, further comprising analyzing acetylation of the MSMB gene histones for distinguishing between an androgen-independent or androgen-refractory prostate cancer and/or an androgen-sensitive prostate cancer.
41. A nucleic acid molecule consisting essentially of a sequence at least 16 continuous bases in length of a sequence selected from the sequence group consisting of SEQ ID NOS: 6 and 8.
Description:
CROSS-REFERENCE TO RELATED APPLICATION
[0001]This application claims the benefit, under 35 U.S.C. § 119(e) to U.S. Provisional Patent Appln. Ser. No. 61/011,537, filed Jan. 18, 2008, the contents of the entirety of which are incorporated herein by this reference.
STATEMENT ACCORDING TO 37 C.F.R. §1.52(e)(5)--SEQUENCE LISTING SUBMITTED ON COMPACT DISC
[0002]Pursuant to 37 C.F.R. §1.52(e)(1)(ii), a compact disc containing an electronic version of the Sequence Listing has been submitted concomitant with this application, the contents of which are hereby incorporated by reference. A second compact disc is submitted and is an identical copy of the first compact disc. The discs are labeled "copy 1" and "copy 2," respectively, and each disc contains one file entitled "Sequence_listing.txt" which is 174 KB and created on Jan. 20, 2009.
TECHNICAL FIELD
[0003]The invention generally relates to medicine and biotechnology.
BACKGROUND
[0004]Several documents are cited throughout the text of this specification. Each of the documents herein (including any manufacturer's specifications, instructions etc.) are hereby incorporated by reference; however, there is no admission that any document cited is indeed prior art of the invention.
[0005]Prostate cancer (PrCa) is the second most common malignancy in males worldwide after lung cancer, and the third leading cause of cancer death in men. Early detection greatly improves survival rates. If the malignant prostate tumor is still local, PrCa can be successfully treated by radiation therapy, surgery, hormone therapy and/or chemotherapy.
[0006]Unfortunately, if the PrCa invades other parts of the body like bones, lymph nodes, rectum and bladder (metastatic PrCa), it becomes refractory to hormone therapy. For this advanced PrCa the prognosis is poor. Currently, PrCa is detected by an elevated level of Prostate-Specific Antigen (PSA) in the blood, along with a digital rectal exam. The PSA test is also used to monitor patients for the recurrence of PrCa following surgery or other treatments. However, although the PSA test has greatly improved the detection of PrCa, its usefulness is still controversial. A recent study by Concato et al. shows that PSA screening is not associated with lower mortality (J. Concato et al. (2006), Arch. Intern. Med. 166:38-43). Moreover, the serum PSA level is also elevated in non-cancerous prostate disorders such as benign prostate hyperplasia and infection.
[0007]Initial tests for suspected prostate cancer are done by analysis of blood levels of protein like PSA or, for instance, PSP94 protein (under development). Positive tests are followed by a conformational diagnosis. The only test which can fully confirm the diagnosis of prostate cancer is a biopsy, the removal of small pieces of the prostate for microscopic examination. The invention provides a novel diagnostic test of prostatic tissue or cells obtainable from prostatic tissue.
[0008]A condition of benign prostatic hyperplasia (BPH) or benign prostatic hypertrophy is common as a man ages. It is thus very important to distinguish between a PrCa and a BPH. Moreover hormone-refractory prostate cancers are more aggressive and need specific treatments such as apoptosis and regression induction of the tumors and/or antimetastasis.
[0009]The latter type of cancer will remain localized in a person's lifetime and is unlikely to reduce life expectancy. In contrast, an aggressive cancer is more lethal, due to metastasis, and requires immediate intervention. Therefore, there is an unmet need for a reliable diagnostic assay and biomarker to distinguish between these two types. The invention provides such.
[0010]PrCa can be distinguished from BPH by diagnosis of prostate cells or tissues, for instance, from prostate tissue biopsy or prostate cells in seminal fluids by assessing the DNA methylation status of non-CpGs (in particular, of CpA and CpT) upstream of or in the promoter region of the MSMB gene as novel biomarker to diagnose for prostate cancer.
[0011]PrCa diagnosis is based on newly identified DNA-methylation markers in the DNA methylome of the MSMB gene and, in particular, the DNA methylation status of specific CpG islands upstream of or in the promoter region of the MSMB gene as novel biomarker.
[0012]The CpG methylation status of specific CpG islands upstream or in the promoter region of the MSMB gene can be used to diagnose for prostate cancer and to distinguish between hormone-refractory and hormone-sensitive prostate cancer, in particular between an androgen hormone-refractory prostate cancer and an androgen hormone-sensitive prostate cancer. Moreover the methylation status of the non CpGs upstream of or in the promoter region of the MSMB gene can be used to distinguish between benign prostate hyperplasia (BPH) and prostate cancer.
[0013]The MSMB gene has a role as an autocrine paracrine factor in uterine, breast and other female reproductive tissues (M. Baijal-Gupta et al., J. Endocrinol. 2000 May, 165(2):425-33). Transcriptional silencing of the MSMB gene is known to be associated with prostate-cancer progression. Expression profiling revealed that the expression of the MSMB gene gradually decreases during the development of PrCa, i.e., from primary PrCa to the late, highly invasive, androgen-independent state (E. S. LaTulippe et al. (2002), Cancer Res. 62:4499-506; D. K. Vanaja et al. (2003), Cancer Res. 63:3877-82; and M. Stanbrough et al. (2006), Cancer Res. 66:2815-25). Actually, the MSMB gene was the most down-regulated of all genes and its expression level decreased about 100-fold in metastatic prostate tissue, as compared to its expression in adjacent benign tissue (D. K. Vanaja et al. (2003), Cancer Res. 63:3877-82).
SUMMARY OF THE INVENTION
[0014]This invention relates generally to the identification of a prostate cell proliferative disorder and method and a diagnostic assay to distinguish between benign prostate hyperplasia (BPH) and prostate cancer. Moreover it relates to a diagnostic assay or method to distinguish between a hormone-sensitive and a hormone-refractory prostate cell proliferative disorder. More specifically this invention relates to analyzing the methylation status of non CpG dinucleotides in the regulatory region surrounding the transcription start site (TSS) of a beta-microseminoprotein (MSMB) gene to distinguish between BPH and prostate cancer. Furthermore the methylation status of CpG dinucleotides regulatory region surrounding the transcription start site (TSS) of a beta-microseminoprotein (MSMB) gene has been proven to provide a power to distinguish between a hormone-refractory prostate cancer (aggressive) and a hormone-sensitive prostate cancer. This is also part of the invention. Both diagnostic assays and methods can be combined to diagnose (together or in sequence) for the diseased status of the prostate. Moreover the invention provides methods and compositions for the diagnosis and for deciding the proper treatment of such prostate cellular proliferation disorders.
[0015]In a particular embodiment this invention relates to a diagnosis for a prostate proliferation disorder specifically by identification hypermethylations of non CpG dinucleotides (for instance, hypermethylation of CpA, CpT or CpC dinucleotides) in particular in a regulatory region surrounding the transcriptional start site of the beta-microseminoprotein (MSMB) gene or in particular upstream of the promoter region or in the promoter region of the beta-microseminoprotein (MSMB) gene. Such diagnosis on cell or tissue samples of the prostate allows specific distinguishing between tissues of benign prostate hyperplasia and prostate cancer.
[0016]More specifically, this invention also relates to a diagnosis for a prostate proliferation disorder specifically by identification hypermethylations in genomic regions that contain a high frequency of CG dinucleotides (CpG islands) and in particular in a regulatory region surrounding the transcriptional start site of the beta-microseminoprotein (MSMB) gene. Hypermethylations in genomic regions that contain a high frequency of CG dinucleotides (CpG islands) and in particular upstream of the promoter region or in the promoter region of the beta-microseminoprotein (MSMB) gene are indicative of a prostate-proliferative disorder that allows to distinguish between a hormone-sensitive and a hormone-refractory prostate cell proliferative disorder.
[0017]A more particular aspect of the invention relates to a diagnostic indicator of an androgen hormone-refractory prostatic tissue cellular proliferative disorder, for instance, an androgen hormone-refractory prostate cancer (PrCa).
[0018]Another aspect of the invention relates to a diagnostic indicator of 1) a benign prostate hyperplasia or a prostate cancer and 2) in case of a prostate cancer of an androgen hormone-sensitive prostatic tissue cancer or an androgen hormone-refractory prostatic tissue cancer.
[0019]The invention allows one to distinguish between hormone-refractory and hormones-sensitive cancer, particularly in prostatic tissues or cells originating from prostatic tissues. However, the test could also be used on body fluids.
[0020]The invention is broadly drawn to methods and assays for detecting a prostate proliferative disorder, in particular for identifying prostate tumor cells that have become refractory or resistant to hormone therapy, and thus allowing to identify the prostate cancer or/and to distinguish hormone-sensitive from hormone-refractory prostate cancers.
[0021]The invention relates generally to the identification of the distinguishing difference between a hormone-refractory prostate tissue cellular proliferative disorder and a hormone-sensitive prostate tissue cellular proliferative disorder in a subject, preferably a human subject. The distinguishing difference relies on the identification of one or more hypermethylated CpG islands surrounding the transcription start site (TSS) of the human gene for beta-microseminoprotein (MSMB), more in particular hypermethylated CpG islands are found in regions upstream of the TSS or in the promoter region of the human gene for beta-microseminoprotein (MSMB).
[0022]The prognostic methods that detect whether a prostate cancer in subjects, preferably human, comprises an androgen-refractory cancer and/or an androgen-sensitive cancer can be carried by biopsy and analysis of the hypermethylation status of the MSMB gene.
[0023]Thus, in a first aspect, the invention provides methods for detecting in a subject of prostate cell proliferative disorder, which methods comprise the steps of: [0024](a) obtaining a biological sample from the subject; [0025](b) determining the methylation state of CpG island upstream and/or downstream of the TSS and/or in the promoter region of the MSMB gene, for instance, the CpG islands about 3.5 kb upstream and about 2.2 kb downstream of the transcriptional start site in the MSMB gene in the subject's sample; and wherein detection of hypermethylation is indicative of a predisposition to, or the incidence of, prostate cancer.
[0026]In a similar aspect, the invention provides methods for detecting in a subject an androgen-refractory prostate cancer, which methods comprise the steps of: [0027](a) obtaining a biological sample from the subject; [0028](b) determining the methylation state of CpG island upstream and/or downstream of the TSS region and/or in the promoter region of the MSMB gene, for instance, the CpG island about 3 kb upstream and about 2.2 kb downstream of the transcriptional start site in the MSMB gene in the subject's sample; and wherein detection of hypermethylation is indicative of a predisposition to, or the incidence of, androgen-sensitive prostate cancer.
[0029]Preferably, both the methods of the invention comprise a further step as follows: [0030](c) identifying hypermethylation of region(s), wherein hypermethylation is identified as being different when compared to the same region(s) of the gene or associated regulatory region in a subject having an androgen-sensitive prostate cancer.
[0031]Another aspect of the invention is that it provides methylation conditions of regulatory regions of the MSMB gene, such as in the CpG islands surrounding the TSS of the human gene for beta-microseminoprotein (MSMB), which can be used (a) to analyze the presence of cancer cells in prostate tissue and/or in prostatic secretions, for instance, in seminal plasma, (b) to define patients that have a prostate cancer or alternatively patients that have a normal prostate, and (c) to define which patients with a prostate cancer have an androgen-refractory prostate cancer or alternatively to define which patients with a prostate cancer have a hormone-sensitive prostate cancer.
[0032]Such test provides an accurate means or tool to decide about the suitable treatment of the prostate cancer; in particular if the MSMB gene is methylated/hypermethylated the need for chemotherapy, surgery or radiation therapy is identified. The methods of the invention can also be used to predict effectiveness of such chemotherapies applicable on a prostate cancer.
[0033]Patients affected by a condition of hypermethylation of regulatory regions of the MSMB gene such as in the CpG islands surrounding the TSS of the MSMB gene, and/or CpG islands upstream of the TSS or in the promoter region of the MSMB gene can, for instance, be treated by DNA methyltransferase (DNMT) inhibitors or can be treated with inhibitors of the EZH2 gene expression or inhibitors of the function of the polycomb protein EZH2 to induce a repair of abnormal methylation.
[0034]Still another aspect of the invention relates to the observation that due to the fact that the MSMB gene, which encodes PSP94 (beta-microsemenoprotein or beta-inhibin), a prostatic secretory protein of 94 amino acids, or PSP57 (lacking an internal exon of 106 bases in the coding region resulting in a frameshift at the 3' end, compared to PSP94) is repressed in hormone-refractory cancer cells, by the hypermethylation of a CpG island in the regulatory regions surrounding the transcriptional start site of the MSMB gene or in the promoter region that the encoding by the MSMB gene or expression of PSP94, known to be a suppressor of tumor growth and metastasis and to be secreted by the prostate gland and functions, is lost in advanced hormone-refractory cancer, for instance, advanced hormone-refractory prostate cancer. PSP57 mRNA is in prostate tumor cell lines, aberrantly spliced and localized in the nuclear fraction of the cell. (J. W. Xuan et al., Oncogene 1995 Sep. 21, 11(6):1041-7.) PSP57 mRNA has been also detected in other urogenital tissues (kidney, bladder) and in most tumor cell lines tested, but was not detectable in other tissues such as breast and lung. (R. Hoffmann et al., Nature Genetics 36:664 (2004).)
[0035]Hypermethylation can be detected by restriction endonuclease treatment and Southern blot analysis. Therefore, in a method of the invention, when the cellular component detected is DNA, restriction endonuclease analysis is preferable to detect hypermethylation of the MSMB regulatory region, in the promoter or upstream of the promoter. Any restriction endonuclease that includes CG as part of its recognition site and that is inhibited when the C is methylated can be utilized. Preferably, the methylation-sensitive restriction endonuclease is BssHII, MspI, or HpaII, used alone or in combination. Other methylation-sensitive restriction endonucleases will be known to those of skill in the art.
[0036]Additional indicators can be part of the diagnostic method of the invention; Moreover the MSMB gene can in androgen-refractory prostate cancer cells, but not in androgen-sensitive prostate cancer cells be trimethylated on histone H3 K27 and the MSMB can be additionally repressed in androgen-refractory prostate cancer cells by the hypoacetylation of H3K9. Assaying for this trimethylation status or this hypoacetylation status can be an additional part of the diagnostic assay or the diagnostic method to distinguish between an androgen-refractory and an androgen-sensitive prostate cancer.
[0037]By the invention, MSMB has been demonstrated and validated to be a true target for repression by the histone methyltransferase EZH2. The identification of MSMB as an EZH2 target gene can explain why the expression of this tumor suppressor gene is lost in advanced stages of prostate cancer. We demonstrated that the increased expression of EZH2 in metastatic prostate cancer results in H3K27 trimethylation of the MSMB gene. This leads to the recruitment of the PRC1 complex and MSMB silencing. In addition, EZH2 binds to DNA methyltransferases and, indirectly, histone deacetylases and these enzymes also contribute to the maintenance of MSMB silencing. Our data demonstrate that specific inhibitors of EZH2 are useful for the treatment of metastatic prostate cancer, at least in part because such inhibitors are expected to reverse the down-regulation of the tumor suppressor PSP94.
[0038]Furthermore, the invention relates generally to the demonstration that the expression of the tumor suppressor PSP94 by MSMB is silenced by EZH2 in advanced prostate cancer cells and that an increased expression of the polycomb protein EZH2 (enhancer of zeste homolog 2), represses transcription via trimethylation of histone H3 on Lys27 (H3K27). The RNAi-mediated knockdown of EZH2 resulted in a loss of H3K27 trimethylation and an increased expression of the MSMB gene. Conversely, the overexpression of EZH2 was associated with a decreased expression of the MSMB gene. PSP94, for prostatic secretory protein of 94 amino acids, is secreted by the prostate gland and functions as a suppressor of tumor growth and metastasis. The expression of PSP94 is lost in advanced, hormone-refractory prostate cancer. Present invention now demonstrates that this decrease of PSP94 expression correlates with an increased expression of the polycomb protein EZH2 (enhancer of zeste homolog 2), which represses transcription via trimethylation of histone H3 on Lys27 (H3K27) and that these events are causally related and that the MSMB gene, which encodes PSP94, is trimethylated on H3K27 in androgen-refractory, but not in androgen-sensitive prostate cancer cells.
[0039]By the invention, it has been demonstrated and validated that the gene encoding the prostatic tumor suppressor PSP94 is a target for repression by the polycomb group protein EZH2. For instance, chromatin immunoprecipitation experiments confirmed an association of EZH2 with the MSMB gene. The RNAi-mediated knockdown of EZH2 resulted in a loss of H3K27 trimethylation and an increased expression of the MSMB gene. Conversely, the overexpression of EZH2 was associated with a decreased expression of the MSMB gene. We also demonstrate that MSMB is additionally repressed in androgen-refractory prostate cancer cells by the hypoacetylation of histone H3K9 and the hypermethylation of a CpG island in the promoter region. Present invention demonstrates a hitherto unexplored link between the putative oncogene EZH2 and the tumor suppressor PSP94, and show that MSMB is silenced by EZH2 in advanced prostate cancer cells.
[0040]The diagnostic method and assay of the invention is thus indicative for the suitability of a treatment of a prostate cell proliferation disorder and in particular a prostate cancer, for instance, an anti-EZH2 treatment by a therapeutically effective amount of an EZH2 inhibitor to prevent the transition of an androgen-refractory prostate cancer to an androgen insensitive prostate cancer. Anti-EZH2 treatment in the art is, for instance, EZH2 siRNA, for instance, the Small interfering RNA (siRNA) duplexes4 targeted against EZH2 reduce the amounts of EZH2 protein present in prostate cells (S. M. Elbashir et al., Nature 411:494-498 (2001)).
[0041]Diagnosis of hypermethylation of the CpG island in the regions surrounding the TSS or in the promoter of the MSMB gene and preferably about 3 kb upstream of the transcriptional start site in the MSMB gene can thus be used as a decision toll for treatment of a patient affected with such hypermethylation with a therapeutically effective amount of an DNA methyltransferase (DNMT) inhibitor for treating the prostate cancer or for preventing that a androgen sensible prostate cancer evolves into an androgen-refractory prostate cancer. MGI Pharma developed small molecule DNA methyltransferase (DNMT) inhibitors for the treatment of cancer. Short oligonucleotide DNA methylation inhibitors in the art are Decitabine 5-Aza-CdR, S110 AzapG, S53 GpAza, S54 GpAzapG, S55 AzapGpAzapG, S56 pGpAzapAzapG, S52R AzapsG, Zebularine and S112 HEGpAzapG. A specific DNMT inhibitor is, for instance, the compound with the structure D in FIG. 6 called S110 or S110 of the company SuperGen which is a dinucleotide containing decitabine, S110, which has superior activity due to increased stability because of less degradation by hydrolytic cleavage and deamination. This is a DNA demethylating agent with a similar activity as decitabine (5-aza-2'-deoxycytidine) or its derivatives. Decitabine is a potent DNA methylation inhibitor which is approved in the US for the treatment of myelodysplastic syndromes (D. B. Yoo et al., Cancer Research 67:6400-6408, No. 13, 1 Jul. 2007). Another DNA methyltransferase (DNMT) inhibitor is MG 98 (HYB 101584) is described in U.S. Pat. No. 6,953,783 and U.S. Pat. No. 6,506,735. MG 98 is a second generation antisense oligonucleotide that selectively targets DNA methyltransferase 1 (DNMT1) mRNA. By inhibiting the production of DNMT, the methylation of DNA is reversed and leads to re-expression of the tumor suppression genes. MG 98 is created by MethylGene Inc. (D. Stewart et al., 11th NCI-EORTC-AACR symposium on new drugs in cancer therapy, 148, 7 Nov. 2000; E. Winquist et al., European Journal of Cancer 38 (Suppl. 7):141, Nov. 2002; D. J. Stewart et al., Annals of Oncology 14:766-774, May 2003; S. Ramchandani et al., Proceedings of the National Academy of Sciences of the United States of America 94:684-689, January 1997; and A. J. Davis et al., 11th NCI-EORTC-AACR symposium on new drugs in cancer therapy, 94, 7 Nov. 2000. These compounds can be administered in a therapeutically efficient amount to patients that have been identified by the diagnostic method of the invention to be in need thereof.
[0042]Thus, epigenetic loss of gene function due to hypermethylation can be rescued by the use of DNA demethylating agents and/or DNA methyltransferase inhibitors and/or HDAC inhibitors. Accordingly, the invention also provides for a method for predicting the likelihood of successful treatment of prostate proliferative disorder or prostate cancer, with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor comprising detecting a methylation change in the region surrounding the TSS or the promotor region of the MBMS gene wherein detection of the methylation change is indicative of successful treatment to a higher degree than if the methylation modification is not detected.
[0043]Also provided is a kit for detecting a predisposition to, or the incidence of, prostate cancer in a sample comprising: [0044](a) means for detecting a methylation change in the region surrounding the TSS or the promotor region of the MBMS gene [0045](b) means for processing a sample derived from the prostate.
[0046]In certain embodiments, a method of diagnosing a disease state or cell proliferative disorder in the prostate of a subject, the method comprising: (a) analyzing the level DNA methylation of regulatory region surrounding the transcription start site (TSS) of a beta-microseminoprotein (MSMB) gene or a homologous sequence in a biological sample isolated from the subject, and (b) comparing the DNA methylation with the DNA methylation in a control sample and/or a benign prostate hyperplasia sample; whereby non-CpG methylation or increased non-CpG methylation relative to the control sample or the benign prostate hyperplasia sample in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer and whereby methylation of the CpG dinucleotides or an increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to a control sample or relative to the benign prostate hyperplasia sample is an indication of a hormone-refractory prostate cancer, androgen-independent prostate cancer (AIPC) or androgen-independent metastatic prostate cancer.
[0047]In certain embodiments, disclosed is a method of diagnosing a disease state or cell proliferative disorder in the prostate in a subject, the method comprising: (a) analyzing the level DNA methylation of the CpG island in the promoter and upstream of the promoter of a beta-microseminoprotein (MSMB) gene or a homologous sequence in a biological sample isolated from the subject, and (b) comparing the DNA methylation with the DNA methylation in a control sample and/or a benign prostate hyperplasia sample; whereby non-CpG methylation or increased non-CpG methylation relative to the control sample or the benign prostate hyperplasia sample in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer and whereby methylation of the CpG dinucleotides or an increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to a control sample or relative to the benign prostate hyperplasia sample is an indication of a hormone-refractory prostate cancer, androgen-independent prostate cancer (AIPC) or androgen-independent metastatic prostate cancer.
[0048]In certain embodiments, disclosed is a method of diagnosing a disease state or cell proliferative disorder in the prostate in a subject, the method comprising: (a) analyzing the level DNA methylation of the transcriptional start site (TSS) of the beta-microseminoprotein (MSMB) gene or a homologous sequence, in particular in regions upstream the TSS or in its promoter region, in a biological sample isolated from the subject, and (b) comparing the DNA methylation with the DNA methylation in a control sample and/or a benign prostate hyperplasia sample; whereby non-CpG methylation or increased non-CpG methylation relative to the control sample or the benign prostate hyperplasia sample in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer and whereby methylation of the CpG dinucleotides or an increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to a control sample or relative to the benign prostate hyperplasia sample is an indication of a hormone-refractory prostate cancer, androgen-independent prostate cancer (AIPC) or androgen-independent metastatic prostate cancer.
[0049]In certain embodiments, a method of diagnosing a disease state or cell proliferative disorder in the prostate in a subject, the method comprising: (a) analyzing the level DNA methylation of regulatory regions surrounding the transcriptional start site of the beta-microseminoprotein (MSMB) gene in a biological sample isolated from the subject, and (b) comparing the DNA methylation with the DNA methylation in a control sample and/or a benign prostate hyperplasia sample; whereby non-CpG methylation or increased non-CpG methylation relative to the control sample or the benign prostate hyperplasia sample in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer and whereby methylation of the CpG dinucleotides or an increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to a control sample or relative to the benign prostate hyperplasia sample is an indication of an androgen-independent metastatic prostate cancer.
[0050]In certain embodiments, a method of diagnosing a disease state or cell proliferative disorder in the prostate in a subject, the method comprising: (a) analyzing the level DNA methylation in the CpG4-5 region of the MSMB gene of a beta-microseminoprotein (MSMB) gene or a homologous sequence in a biological sample isolated from the subject, and (b) comparing the DNA methylation with the DNA methylation in a control sample and/or a benign prostate hyperplasia sample; whereby non-CpG methylation or increased non-CpG methylation relative to the control sample or the benign prostate hyperplasia sample in the regulatory regions surrounding the transcriptional start site of the MSMB gene is an indication for prostate cancer and whereby methylation of the CpG dinucleotides or an increased level methylation of the CpG dinucleotides in the regulatory region surrounding the transcriptional start site of the MSMB gene relative to a control sample or relative to the benign prostate hyperplasia sample is an indication of a hormone-refractory prostate cancer, androgen-independent prostate cancer (AIPC) or androgen-independent metastatic prostate cancer.
[0051]Further specific embodiments of these previous methods of diagnosis can be:
[0052]The previous method of diagnosing further comprising a step of analyzing histone (de)acetylation of the MSMB gene in the sample.
[0053]The previous method of diagnosing whereby the disease state or cell proliferative disorder is a cancer.
[0054]The previous method of diagnosing to distinguish between a healthy prostate and a disordered or diseased prostate.
[0055]The previous method of diagnosing to distinguish between a benign prostate hyperplasia and a prostate cancer.
[0056]The previous method of diagnosing to distinguish between a hormone-sensitive prostate cancer and a hormone-refractory prostate cancer.
[0057]The previous method of diagnosing to distinguish between an androgen-sensitive prostate cancer or androgen dependent prostate cancer and androgen-independent prostate cancer (AIPC).
[0058]The previous method of diagnosing to discover an androgen-independent metastatic prostate cancer in a prostate cell or prostate tissue.
[0059]The previous method of diagnosing to carry out a prostate cancer grading or prostate cancer staging.
[0060]The previous method of diagnosing to decide on the proper treatment or proper medicament of the prostate disease state.
[0061]The previous method of diagnosing to decide on the treatment with a pharmaceutically acceptable DNA methylation inhibitor.
[0062]The previous method of diagnosing to decide on the treatment with a pharmaceutically acceptable HDAC inhibitor.
[0063]The previous method of diagnosing to decide on the treatment to decrease the activity of the EZH2 protein.
[0064]The previous method of diagnosing to decide on the treatment with a DNA demethylating agent and/or a DNA methyltransferase inhibitor and/or HDAC inhibitor.
[0065]The previous method of diagnosing to decide on a prophylactically effective amount of a nutraceutical. To treat a subject with a prostate disease status.
[0066]Further scope of applicability of the invention will become apparent from the detailed description given hereinafter. However, it should be understood that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.
DESCRIPTION OF THE DRAWINGS
[0067]FIG. 1 demonstrates MSMB and EZH2 expression in prostatic epithelial cells and trimethylation of the MSMB gene on H3K27. (a) The relative amounts of EZH2 (left panel) and MSMB transcripts (right panel) in the indicated prostate cell lines were determined by quantitative RT-PCR with intron-spanning primers. Human prostate PC-3 cells (adenocarcinoma), LNCaP cells (carcinoma) and DU 145 cells (carcinoma) were cultured as monolayers in 50% Dulbecco's modified Eagle's medium (DMEM) and 50% Ham's F12, RPMI1640 and DMEM, respectively, supplemented with 10% fetal calf serum. PZ-HPV-7 cells, an immortalized cell line derived from normal human prostate cells, were cultured in keratinocyte-serum free medium supplemented with 5 ng/ml human recombinant epidermal growth factor and 0.05 mg/ml bovine pituitary extract. Total RNA was isolated using the Genelute Mammalian Total RNA Miniprep kit (Sigma, St. Louis, Mo., USA). A total of 1-5 mg RNA was reverse-transcribed with oligo dT primer (Sigma) and the M-MulV reverse transcriptase (Fermentas GMBH, St. Leon-Rot, Germany). cDNA (1.5%) was analyzed by real-time PCR in triplicate using a Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen, Paisly, UK) in a Rotorgene detection system (Corbett Research, Cambridge, UK) and normalized to the housekeeping gene hypoxanthine-guanine-phosphoribosyl-transferase (HPRT). All used primer sequences are available on request. The data represent the means±s.e. of at least three independent experiments. (b) Schematic representation of the MSMB gene on scale. The four exons are indicated by the black boxes. The black lines below the MSMB locus represent the fragments (1-5) amplified by ChIP analysis. (c) ChIPs on PC-3 (left panel) and LNCaP (right panel) cells were performed with 10 μg of control antibodies (rabbit anti-mouse IgGs, Dakocytomation, Gostrup, Denmark) or 5 μg of antibodies against H3K27me3 (polyclonal anti-H3K27, Upstate, Dundee, UK). ChIP reactions were performed according to the protocol of Upstate, Dundee, UK. The DNA was recovered with the Genelute PCR clean-up kit (Sigma) and analyzed by real-time PCR. Numbers 1-5 refer to the MSMB fragments that were amplified (see panel b). The data represent the means±s.e. of at least three independent experiments in duplicate and indicate the fold enrichment as compared to the negative control (IgGs). The enrichment of DNA was calculated using the formula:fold enrichment=2(CtlgG-CtAb), where Ct is the threshold cycle, IgG is the normal rabbit IgG and Ab is the specific antibody.
[0068]FIG. 2 concerns how EZH2 is recruited to the MSMB gene and causes trimethylation of H3K27. (a) ChIP assays were performed in PC-3 cells using rabbit antibodies against a synthetic peptide of human EZH2 (E. Vire et al. (2006), Nature 439:871-874), trimethylated H3K27 (Upstate), trimethylated H3K9 (Upstate) and control IgGs. ChIP results were revealed by EtBr staining of agarose gels containing PCR-amplified ChIP DNA. (b) Immunoblotting (upper panel) and quantitative RT-PCR analysis (lower panel) of PC-3 cell lysates, obtained after transfection with either a control siRNA (Ctr), that is, a scrambled version of a siRNA duplex for the housekeeping gene PPP1R8 (GGAACUCGAACCUCCACGAACAAUU (SEQ ID NO:71), Invitrogen) or an EZH2 siRNA (KD; AAGACUCUGAAUGCAGUUGCU (SEQ ID NO:72), Dharmacon, Chicago, Ill., USA). The PC-3 cells were plated at 1.2×106 cells in a 10 cm plate. At 24 hours after plating, the cells were transfected with 300 nM of siRNA duplex using Lipofectamine 2000 (Invitrogen). At 48 hours after transfection, the cells were harvested. SIPP1 served as a loading control for the immunoblotting and ACTIN was used as control for normalization in the quantitative RT-PCR. (c and d) ChIP assays were performed on chromatin obtained from PC-3 cells transfected with either control (Ctr) or EZH2 siRNAs, using control antibodies (rabbit IgGs), antibodies against EZH2 (c) or antibodies against H3K27me3 (d). The immunoprecipitated DNA was analyzed by quantitative PCR using primers specific for the MSMB gene (FIG. 1, Panel b). The enrichment on MSMB is expressed as a %±s.e. of the control value (n=3-4). (e) PC-3 cells were transfected with control (A lamin A/C siRNA duplex (AACUGGACUUCCAGAAGAACA (SEQ ID NO:73), Dharmacon) or EZH2 siRNAs for 48 hours. The steady-state transcript levels of MSMB, EZH2 and the housekeeping gene PPP1R8 were determined by quantitative RT-PCR analysis with intron-spanning primers specific for the indicated genes and were expressed relative to the transcript level in the control condition. ACTIN was used as a control for normalization. (f) PZ-HPV7 cells were transiently transfected with an expression vector encoding either Gal4-tag alone or its fusion with EZH2 using lipofectamine plus (Invitrogen), according to manufacturer's instructions. The analysis was carried out as described in (e), with the housekeeping gene HPRT as negative control.
[0069]FIG. 3 demonstrates that the MSMB gene is regulated by histone (de)acetylation. (a) PC-3 cells were treated for nine hours with 50 ng/ml of the histone deacetylase inhibitor trichostatin A (TSA, Sigma). Subsequently, the steady-state levels of the MSMB and EZH2 transcripts were determined by quantitative RT-PCR analysis with intron-spanning primers specific for the indicated genes. The data are expressed relative to the transcript level in the control condition. HPRT was used as a control for normalization. (b) ChIPs on PC-3 (left panel) and LNCaP (right panel) cells were performed with 10 μg of control antibodies (rabbit anti-mouse IgGs, Dakocytomation) or 5 μg of antibodies against H3K9ac (polyclonal anti-H3K9ac, Upstate). The data represent the means±s.e. of three independent experiments in duplicate and indicate the fold enrichment as compared to the negative control with IgG.
[0070]FIG. 4 demonstrates that the MSMB gene is regulated by methylation of a CpG island in the promoter region. (a) PC-3 cells were treated for 48 hours with 10 mM of the DNA methyltransferase inhibitor 50-azacytidine. Subsequently, the steady-state levels of the MSMB and EZH2 transcripts were determined by quantitative RT-PCR analysis with intron-spanning primers specific for the indicated genes. The data were expressed relative to the transcript level in the control condition. HPRT was used as a control for normalization. (b) A schematic representation of the MSMB gene on scale. The four exons are indicated by the black boxes and the two analyzed CpG regions by black stars. TSS, transcriptional start site. Methylated CG dinucleotides are denoted underneath by closed circles and unmethylated CGs by open circles. Genomic DNA of PC-3 cells or LNCaP cells was purified with the GenElute Mammalian Genomic DNA Miniprep kit of Sigma. Two microgram was digested overnight with BglII. The DNA was denatured with 0.3 M NaOH at 421° C. for 30 minutes. Sodium bisulfite (3.3 M) and hydroquinone (0.5 mM) were added to the DNA and the mixture was incubated overnight at 55° C.
[0071]The DNA was purified with the PCR purification kit of Sigma, St Louis, Mo., USA. The DNA was desulfonated with 0.3 M NaCl for 15 minutes at 37° C. and precipitated by adding NH4Ac and ethanol. The pellet was air-dried and dissolved in 10 mM Tris and 1 mM ethylene-diaminete-traacetic acid at pH 8. PCR was performed with Jumpstart Taq Polymerase (Sigma). The primers GTTTAGGTTGGAGTGTAGTGG (SEQ ID NO:74) (sense) and ATCCTAACTAACATAATAAAACCCC (SEQ ID NO:75) (antisense) were used to amplify the first CpG island and the primers AGTTTTTTTATTTAGGGGTGGATTTTA (SEQ ID NO:76) (sense) and CCAAACTAATCTCAAATACCTAACCTC (SEQ ID NO:77) (antisense) were used to amplify the second CpG island. The PCR products were subcloned in the pGem-T vector of Promega according to the manufacturer's protocol. At least ten clones for each condition were sequenced. The plasmids were sequenced on a MegaBace sequencer. The percentages of methylated CpG dinucleotides are indicated in the bar diagrams.
[0072]FIG. 5 displays chemical structures of DNA methylation inhibitors: 5-aza-CdR (A), S52 (B), S53 (C), S110 (D), and S112 (E). 5-Aza-CdR is a deoxycytidine with an extra nitrogen at the 5-position of the pyrimidine ring. S52 is a phosphorothioate analogue of S110. There are two optical isomers of S52: S52S and S52R. S53 is a dinucleotide with a guanosine at the 5'-end and 5-aza-CdR at the 3'-end. S110 is a reverse dinucleotide of S53 containing a 5-aza-CdR at the 5'-end followed by a guanosine. S112 is a triethylamine salt of 5'-AzapG-3' dinucleotide with a hexaethylene glycol phosphate at the 5'-end.
[0073]FIG. 6 provides a schematic representation of SEQ ID NO:5: 15000 bp upstream and downstream of the transcriptional start site (TSS) (first nucleotide of exon 1) of the MSMB gene. The MSMB gene is located on the forward strand of chromosome 10 from 51219559 to 51232596 with transcriptional start site (TSS)=51219559. The sequence (SEQ ID NO:5) was obtained from the Homo sapiens chromosome 10 genomic contig with the accession number NT--008583.16 (Hs10--8740:85708-115707). All CpG islands, except for CpG5 and 7, are predicted by the program Newcpgreport, with the following parameters: window=50, window shift=1, Island size>200, GC %>0.2 and O/E (observed/predicted>0.2. CpG5 and CpG7 islands are predicted by the program Methprimer with the following parameters: Island size>100, GC %>0.5 and O/E>0.6. The exact location of the CpG islands are described in Table 1. The predicted CpG islands (CpG 1-CpG 10) are shown by Shaded arrows and exons (E1-E4) by shaded boxes.
[0074]SEQ ID NO:5 represents genomic sequences of the forward strand of chromosome 10 from 15000 nt upstream and downstream of the transcriptional start site (TSS) of the MSMB gene and SEQ ID NO:6 represent the bisulfite converted sequence thereof. The TSS of the MSMB gene is located at position 51219559 on the forward strand of chromosome 10.
[0075]SEQ ID NO:7 represents genomic sequences of the reverse strand of chromosome 10 from 15000 nt upstream and downstream of the transcriptional start site (TSS) of the MSMB gene and SEQ ID NO:8 represent the bisulfite converted sequence thereof. The TSS of the MSMB gene is located at position 51219559 on the forward strand of chromosome 10.
DETAILED DESCRIPTION OF THE INVENTION
[0076]The invention demonstrates that the MSMB gene is silenced by DNA methylation of regulatory regions surrounding the transcriptional start site (TSS) of the concerned gene, in particular in regions upstream the TSS or in its promoter region. Novel specific CpG islands have been such as the CpG islands about 3 kb upstream and about 2.2 kb downstream of the transcriptional start site in the MSMB gene and showed by bisulphite sequencing that in the androgen-independent metastatic PrCa PC-3 cells the CpG dinucleotides were methylated.
[0077]Furthermore, we could show that the MSMB gene can be reactivated in PC-3 cells by the addition of the DNA methyltransferase inhibitor 5' azacytidine and that this was associated with a decreased methylation of the upstream CpG island.
[0078]Importantly, the CpG island about 3 kb upstream of the transcriptional start site in the MSMB gene was hypomethylated in the androgen-sensitive LNCaP cells as compared to its methylation status in the androgen-refractory PC-3 cells. This agrees with the higher expression of the MSMB gene in LNCaP cells as compared to its expression in PC-3 cells.
[0079]It has been demonstrated that the methylation of CpG sites is the major factor underlying the transcriptional silencing of the MSMB gene in PrCa cell lines and this suggests that the DNA methylation state of the MSMB gene can be used as a biomarker for PrCa.
[0080]"Disease state" as used herein means any disease, disorder, condition, symptom, or indication.
[0081]As used herein, the term "cell proliferative disorder" refers to conditions in which the unregulated and/or abnormal growth of cells can lead to the development of an unwanted condition or disease, which can be cancerous or non-cancerous. The detection of the cell proliferative disorder may be by way of routine examination, screening for a cell proliferative disorder or pre-stadia such cell proliferative disorder, monitoring and/or staging the state and/or progression of the cell proliferative disorder, assessing for recurrence following treatment, and monitoring the success of a treatment regimen. In certain embodiments, the cell proliferation disorder is cancer.
[0082]As used herein, the term "cancer" as used herein concerns malignant neoplasm, malignant tumor or invasive tumor and also can include solid neoplasm or solid tumors cancers. Cancers are classified by the type of cell that resembles the tumor and, therefore, the tissue presumed to be the origin of the tumor. Examples of general categories include: Carcinoma: Malignant tumors derived from epithelial cells. This group represents the most common cancers, including the common forms of breast, prostate, lung and colon cancer. Sarcoma: Malignant tumors derived from connective tissue, or mesenchymal cells. Lymphoma and leukemia: Malignancies derived from hematopoietic (blood-forming) cells Germ cell tumor: Tumors derived from totipotent cells. In adults most often found in the testicle and ovary; in fetuses, babies, and young children most often found on the body midline, particularly at the tip of the tailbone; in horses most often found at the poll (base of the skull). Blastic tumor: A tumor (usually malignant) which resembles an immature or embryonic tissue.
[0083]"Hormone-refractory prostate cancer" and in particular "androgen-independent prostate cancer (AIPC)" has to be understood for the meaning of this invention as prostate cancer that has become refractory, that is, it no longer responds to hormone therapy.
[0084]"Prostate cancer grading" as used herein means describing how abnormal or aggressive the cancer cells appear. The grade helps to predict long-term results, response to treatment and survival. In the art there is, for instance, the Gleason scale that is the most common scale used for grading prostate cancer. This system assigns cancer cells a score from 1 to 10, by combining the two most common patterns of cells to give a total score (i.e., 3+4=grade 7). Scores generally range between 4 and, most commonly, 6 or 7. These scores are broken down into three main levels: Low-grade (well differentiated): This type of slow-growing cancer has an appearance most like normal prostate cells and is the least dangerous. It has a Gleason score of 4 or less. Intermediate grade (moderately differentiated): This type is somewhere between the low- and high-grade cancers and the most common of the three. Depending on PSA level and tumor volume, it can act like a high- or low-grade cancer. It has Gleason score between 4 and 7. High-grade (poorly differentiated). This type of cancer has an appearance least like normal prostate cells. It is the most deadly since it is very aggressive and grows very fast--even into surrounding areas such as lymph nodes and bones. These cancer cells also tend to be large, hard to treat, and reappear more frequently. They have a Gleason score between 8 and 10 (A. S. Perry et al., Endocrine-Related Cancer 13 (2) 357-377).
[0085]"Prostate cancer staging" as used herein concerns how much and where the cancer is located. The more cancer there is in the body, the more likely it is to spread and less likely that treatments will work. Therefore, the more advanced stages can affect long-term results and survival. According an older prostate cancer staging the prostate cancer is broken down into four primary stages, for instance, the four ABCD stages of staging to gauge the severity of prostate cancer to describe the detection and location of the cancer. Stage A: Cancer found when not suspected or due to a high PSA level, Stage B: Cancer found due to abnormal digital rectal exam and is held in the prostate, Stage C: Cancer that has spread to the tissues outside of the prostate, Stage D: Cancer that has spread to the lymph nodes or bone. A particular system in the art which replaced the ABCD staging system of prostate cancer to give an even more accurate description of the cancer is the TNM grading system. "T" describes the tumor and uses different numbers to explain how large it is; "N" stands for nodes and tells whether the cancer has spread to the lymph nodes; "M" means metastatic, and tells whether the cancer has spread throughout the body. There are various T Status stages: Stage T1: Microscopic tumor confined to prostate and undetectable by a digital rectal exam (DRE) or ultrasound; Stage T1a: Tumor found in 5% or less of prostate tissue sample; Stage T1b: Tumor found in more than 5% of a prostate tissue sample; Stage T1c: Tumor is identified by needle biopsy as a follow-up to screening that detected elevated PSA results; Stage T2: Tumor confined to prostate and can be detected by DRE or ultrasound; Stage T2a: Tumor involves less than half of one lobe of the prostate, and can usually be discovered during DRE exam; Stage T2b: Tumor involves more than half of one lobe of the prostate, and can usually be felt during DRE exam; Stage T2c: Tumor involves both lobes of the prostate and is felt during a DRE exam; Stage T3: Tumor has spread to surrounding tissues or to the seminal vesicles; Stage T3a: Tumor has spread to outside of the prostate on only one side; Stage T3b: Tumor has spread to outside of the prostate on both sides; Stage T3c: Tumor has spread to one or both of the seminal tubes; Stage T4: Tumor is still within the pelvic region but may have spread to organs near the prostate, such as the bladder; Stage T4a: Tumor has spread beyond the prostate to any or all of the bladder neck, the external sphincter, and/or the rectum and Stage T4b: Tumor has spread beyond the prostate and may affect the levator muscles (the muscles that help to raise and lower the organ) and/or the tumor may be attached to the pelvic wall and various N Status stages: Stage N0: Cancer cells have spread, but not yet to pelvic lymph nodes; Stage N1: Cancer cells have spread to a single lymph node in the pelvic area and are 2 cm (approximately 3/4 of one inch) or less in size; Stage N2: Cancer cells have spread either to a single lymph node and are more than 2 cm but less than 5 cm (approximately 2 inches) in size, or the prostate cancer cells are found in more than one lymph node and are no larger than 5 cm in size; Stage N3: Cancer cells have spread to the lymph nodes and are larger than 5 cm in size and various M Status stages: Stage M0: Cancer cells have spread, but only regionally in the pelvic area & Stage M1: Cancer cells have spread beyond the pelvic area to other parts of the body (Dr. F. H. Schroder et al., The Prostate Volume 21, Issue S4, pp. 129-138, 20 Jul. 2006).
[0086]As used herein, the term "effective amount" refers to an amount of a compound, or a combination of compounds, of the invention effective when administered alone or in combination as an anti-proliferative agent. For example, an effective amount refers to an amount of the compound present in a formulation or on a medical device given to a recipient patient or subject sufficient to elicit biological activity, for example, anti-proliferative activity, such as e.g., anti-cancer activity or anti-neoplastic activity. The combination of compounds optionally is a synergistic combination. Synergy, as described, for example, by Chou and Talalay, Adv. Enzyme Regul. vol. 22, pp. 27-55 (1984), occurs when the effect of the compounds when administered in combination is greater than the additive effect of the compounds when administered alone as a single agent. In general, a synergistic effect is most clearly demonstrated at sub-optimal concentrations of the compounds. Synergy can be in terms of lower cytotoxicity, or increased anti-proliferative effect, or some other beneficial effect of the combination compared with the individual components.
[0087]A "therapeutically effective amount" as used herein means the amount of a compound that, when administered to a mammal for treating a disease, is sufficient to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the mammal to be treated. A therapeutically effective amount of one or more of the compounds can be formulated with a pharmaceutically acceptable carrier for administration to a human or an animal. Accordingly, the compounds or the formulations can be administered, for example, via oral, parenteral, or topical routes, to provide an effective amount of the compound. In alternative embodiments, the compounds prepared in accordance with the invention can be used to coat or impregnate a medical device.
[0088]The term "prophylactically effective amount" as used herein means an effective amount of a compound or compounds, of the invention that is administered to prevent or reduce the risk of unwanted cellular proliferation.
[0089]"Pharmacological effect" as used herein encompasses effects produced in the subject that achieve the intended purpose of a therapy. In one preferred embodiment, a pharmacological effect means that primary indications of the subject being treated are prevented, alleviated, or reduced. For example, a pharmacological effect would be one that results in the prevention, alleviation or reduction of primary indications in a treated subject. In another preferred embodiment, a pharmacological effect means that disorders or symptoms of the primary indications of the subject being treated are prevented, alleviated, or reduced. For example, a pharmacological effect would be one that results in the prevention or reduction of primary indications in a treated subject.
[0090]"Prostate biopsy" as used herein is a procedure in which small samples are removed from a man's prostate gland to be tested for the presence of cancer. It is typically performed when the scores from a PSA blood test rise to a level that is associated with the possible presence of prostate cancer.
[0091]"Treating" includes any effect, e.g., lessening, reducing, modulating, or eliminating, that results in the improvement of the condition, disease, disorder, etc. "Treating" or "treatment" of a disease state includes: (1) preventing the disease state, i.e., causing the clinical symptoms of the disease state not to develop in a subject that may be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state; (2) inhibiting the disease state, i.e., arresting the development of the disease state or its clinical symptoms; or (3) relieving the disease state, i.e., causing temporary or permanent regression of the disease state or its clinical symptoms.
[0092]By "homologous sequence" is meant, a nucleotide sequence that is shared by one or more polynucleotide sequences, such as genes, gene transcripts and/or non-coding polynucleotides. For example, a homologous sequence can be a nucleotide sequence that is shared by two or more genes encoding related but different proteins, such as different members of a gene family, different protein epitopes, different protein isoforms or completely divergent genes, such as a cytokine and its corresponding receptors. A homologous sequence can be a nucleotide sequence that is shared by two or more non-coding polynucleotides, such as noncoding DNA or RNA, regulatory sequences, introns, and sites of transcriptional control or regulation. Homologous sequences can also include conserved sequence regions shared by more than one polynucleotide sequence. Homology does not need to be perfect homology (e.g., 100%), as partially homologous sequences are also contemplated by the instant invention (e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).
[0093]With the "regulatory region surrounding the transcription start site (TSS)" is meant a regulatory region located upstream or 5' to the TSS and/or a regulatory region around the TSS and/or a regulatory region located downstream or 3' to the TSS of the concerned gene. The location of the concerned region can vary from 15 Kbp upstream to 15 Kbp downstream of the TSS. Thus the region under investigation may correspond to all or part of the promotor region of the MSMB gene. Alternatively, the region under investigation corresponds with an exon and/or intron region and/or TSS region of the MSMB gene. The up stream region of the MSMB gene is preferably between -13877 and -13583 or between -10528 and -10254 or between -3920 to -3673 or between -3471 and -3141 or -3128 and -2817 or between -3533 and -2734 base pairs from the transcription start site, and/or preferably between -452 bp upstream and +152 bp downstream from the TSS and/or between 1671 and 1996 or between 2180 and 2390 between 5236 and 5616 or between 7670 and 8030 or between 11432 and 11754 base pairs downstream from the transcription start site of the MSMB gene is analyzed for hypermethylation. Most preferably, the region extends from -3128 bp to -2817 bp upstream from the transcription start site of the MSMB gene and/or between -452 bp upstream and +152 bp downstream from the TSS and/or extends from 2180 bp to 2390 bp downstream from the transcription start site of the MSMB gene.
[0094]The term "promoter" refers to the regulatory region located upstream, or 5' to the structural gene and/or TSS. Such a region extends typically between approximately 5 Kb, 500 bp or 150 to 300 bp upstream from the transcription start site of the concerned gene. For the MSMB gene, we identified at least two CpG islands (genomic regions that contain a high frequency of CG dinucleotides) surround the transcriptional start site, by the use of the bioinformatics program Methprimer (parameters: Island size>100 nt, GC %>0.5 and Observed/expected (O/E)>0.6; FIG. 4) but a more extended bioinformatics approach analysis (program Newcpgreport, with parameters: window=50, window shift=1, Island size>200, GC %>0.2 and O/E>0.2) predicted 8 additional islands as shown in Table 1.
[0095]By "conserved sequence region" is meant, a nucleotide sequence of one or more regions in a polynucleotide does not vary significantly between generations or from one biological system, subject, or organism to another biological system, subject, or organism. The polynucleotide can include both coding and non-coding DNA and RNA.
[0096]By "EZH2" as used herein is meant, the EZH2 gene and any polycomb group protein EZH2 protein, peptide, or polypeptide having any polycomb group protein EZH2 activity, such as encoded by EZH2 or any other polycomb group protein EZH2 transcript derived from an EZH2 gene. The term EZH2 also refers to nucleic acid sequences encoding any polycomb group protein EZH2 protein, peptide, or polypeptide having EZH2 activity. The term "EZH2" is also meant to include other EZH2 encoding sequence, such as other EZH2 isoforms, mutant EZH2 genes, splice variants of EZH2 genes, and EZH2 gene polymorphisms. The polycomb group protein enhancer of zeste homolog 2 (EZH2) is overexpressed in hormone-refractory, metastatic prostate cancer (Varambally et al., 2002, Nature 419, 624-629). An example of such EZH2 is, for instance, the unprocessed precursor with entry in the UniProtKB/Swiss-Prot and with primary accession number Q15910, Protein name Enhancer of zeste homolog 2 Synonym ENX-1 Gene name Name: EZH2 and the sequence of the unprocessed precursor (Length: 746 AA (This is the length of the unprocessed precursor) Molecular weight: 85363 Da as demonstrated by SEQ ID NO: 1.
[0097]A published sequence of the EZH2 mRNA is, for instance, Homo sapiens enhancer of zeste homolog 2 (Drosophila) (EZH2), transcript variant 1, mRNA. as published in NMCI with SEQ ID NO:2 and published in G. Laible et al., EMBO J. 16 (11), 3219-3232 (1997); H. Chen et al., Genomics 38 (1), 30-37 (1996); K. J. Abel et al., Genomics 37 (2), 161-171 (1996); and Cardoso et al., European Journal of Human Genetics vol. 8, January 2000, pages 174-180.
[0098]The gene MSMB encodes, microseminoprotein, beta (also known as MSP; PSP; IGBF; MSPB; PN44; PRPS; PSP57; PSP94; PSP-94) which is a member of the immunoglobulin binding factor family. PSP94 encoded by the MSMB gene is a tumor suppressor. The Prostate secretory protein of 94 amino acids (PSP94), encoded by the highly prostate-specific MSMB gene, is one of the three major proteins secreted in the seminal fluid, together with PSA and Prostatic Acid Phosphatase (PAP). It has been shown that PSP94 decreases tumor growth in a syngenic in vivo model of PrCa (et al. (2003) Cancer Res. 63:2072-8) and suppresses PC-3 cell clonogenic growth as well as the growth of PC-3 xenografts (S. V. Garde et al. (1999), Prostate 38:118-25). Interestingly, the peptide PCK3145, derived from PSP94 and patented by Ambrilia (Biopharmaceutical company, Quebec, Canada), is currently being clinically tested for the treatment of advanced PrCa (R. E. Hawkins, L. Daigneault, R. Cowan, R. Griffiths, C. Panchal et al. (2005), Clin. Prostate Cancer 4:91-9).
[0099]The MSMB gene is approximately 13 kb in length, comprises 4 exons and 3 introns, and encodes a transcript of 572 nucleotides (FIG. 6). It is synthesized by the epithelial cells of the prostate gland and secreted into the seminal plasma. This protein has inhibin-like activity. It may have a role as an autocrine paracrine factor. The expression of the encoded protein is found to be decreased in prostate cancer. Two alternatively spliced transcript variants encoding different isoforms are described for this gene. One transcript variant (Homo sapiens microseminoprotein, beta-MSMB, transcript variant PSP94) has been deposited in NCBI under the accession number NM--002443.2 of which the 572 bp mRNA has been described as in SEQ ID NO:3.
[0100]The other variant (Homo sapiens microseminoprotein, beta-(MSMB), transcript variant PSP57, mRNA) has been deposited in CBI under the accession number NM--138634.1 of which the 466 bp mRNA has been described as in SEQ ID NO:4.
[0101]The tumor suppressor PSP94, also known as b-microseminoprotein or prostatic inhibin, is a small (10.7 kDa), non-glycosylated and cysteine-rich protein that is abundantly secreted by the prostate gland and is found in both seminal fluid and blood (S. V. Garde et al. (1999), Prostate 38:118-125; Shukeir et al., 2003; Annahi et al., 2005; and S. Lamy et al. (2006), Int. J. Cancer 118:2350-2358). It is also know that the expression of PSP94 progressively decreases during the development of prostate cancer from an early, low-invasive, androgen-dependent state to a late, highly invasive, androgen-refractory state (E. LaTulippe, J. Satagopan et al. (2002), Cancer Res. 62:4499-4506; D. K. Vanaja et al. (2003), Cancer Res. 63:3877-3882; M. Stanbrough et al. (2006), Cancer Res. 66:2815-2825). The gradual loss of PSP94 is likely to contribute to the development of prostate cancer because PSP94 impedes prostate cancer growth and metastasis (S. V. Garde et al. (1999), Prostate 38:118-125; N. Shukeir, A. Arakelian et al. (2004), Cancer Res. 64:5370-5377; and N. Shukeir et al. (2003), Cancer Res. 63:2072-2078). It is not known how the expression of the PSP94-encoding MSMB gene is regulated.
[0102]The molecular basis for the tumor-suppressor function of PSP94 is complex as this protein has been found to promote tumor cell apoptosis (S. V. Garde et al. (1999), Prostate 38:118-125), to inhibit the secretion of a matrix metalloproteinase that is implicated in tumor metastasis (B. Annahi et al. (2005), Clin. Exp. Mestas. 22:429-439), and to decrease tumor-associated, vascular endothelial growth factor (VEGF)-mediated vascularization (S. Lamy et al. (2006), Int. J. Cancer 118:2350-2358).
[0103]Interestingly, the antitumor effects of PSP94 can be recapitulated with a synthetic peptide comprising an N-terminal fragment of PSP94 and this peptide is currently clinically tested for the treatment of metastatic prostate cancer (S. Lamy et al. (2006), Int. J. Cancer 118:2350-2358).
[0104]As aforementioned, the invention is based on the unexpected discovery that the level of methylation of the MSMB gene promoter and/or the region upstream from the transcription start site of the MSMB gene is different in androgen-sensitive when compared to androgen-refractory prostate cancers. Hypermethylation of the MSMB gene promoter and/or region results in a reduced transcription of MSMB and encoding of PS94 which is regulated by EZH2.
[0105]Accordingly, in a first aspect, provided is a method that identifies a prostate cell proliferative disorder in a human male subject, the method comprises: [0106]providing a sample of prostatic tissue and/or biological fluid of the prostate from a human patient susceptible to a prostate cancer and, [0107]analyzing the sample for the presence of hypermethylation (on CpG and/or non-CpG dinucleotides) in a regulatory region surrounding the transcription start site or promoter region of the MSMB gene, [0108]wherein the presence of hypermethylation in this region is indicative of prostate cancer.
[0109]Preferably, the method comprise the steps of: [0110](a) obtaining a biological sample from the subject; [0111](b) determining the methylation state of CpG island upstream and/or downstream of the TSS region and/or in the promoter region of the MSMB gene, for instance, the CpG islands about 3 kb upstream and about 2.2 kb downstream of the transcriptional start site in the MSMB gene in the subject's sample; and [0112](c) identifying hypermethylation of the region(s), wherein hypermethylation (on CpG and/or non-CpG dinucleotides) is identified as being different when compared to the same region(s) of the gene or associated regulatory region in a subject not having the prostate cellular proliferative disorder, [0113]wherein detection of hypermethylation is indicative of a predisposition to, or the incidence of, prostate cancer.
[0114]The sample for use in such methods is preferably a tissue sample. Prostate biopsy is a procedure in which small samples are removed from a man's prostate gland to be tested for the presence of cancer. It is typically performed when the scores from a PSA blood test rise to a level that is associated with the possible presence of prostate cancer. A biopsy thus provides a specific example of a biological sample for use in present methods. Examination of the condition of the prostate may be performed transrectally, through the ureter or through the perineum. The most common procedure is transrectal, and may be done with tactile finger guidance (M. Ghei, S. Pericleous, et al. (2005 September), Ann. R. Coll. Surg. Engl. 87 (5):386-7) or with ultrasound guidance. If cancer is suspected, a biopsy is offered. During a biopsy tissue samples from the prostate are obtained, for instance, via the rectum. A biopsy gun can be used to insert and remove special hollow-core needles (usually three to six on each side of the prostate) in less than a second.
[0115]Suitable samples for diagnostic, prognostic, or personalized medicinal uses can be obtained from surgical samples, such as biopsies or surgical resection. However, other suitable samples for use in the methods of the invention comprise fine needle aspirates, paraffin embedded tissues, frozen tumor tissue samples, fresh tumor tissue samples, fresh or frozen body fluid. Examples of body fluids include prostatic fluids, blood samples, serum, plasma, urine, ejaculate, wash or lavage fluid. In fact, any tissue or fluid containing cells or nucleic acid, preferably DNA, derived from cells of the prostate is a suitable reagent for use in the methods of the invention. Present methods preferably also include the step of obtaining the suitable sample. Cells may need to be lysed for release of the nucleic acid. The nucleic acid may need to be cleared of proteins or other contaminants, e.g., by treatment with enzymes. The nucleic acid may also need to be concentrated prior to further use in the method of the invention, in particular when the nucleic acid is derived from bodily fluids.
[0116]As shown herein, the above mentioned methods for identifying prostate tumor cells also allow distinguishing hormone-sensitive from hormone-refractory prostate cancers.
[0117]Thus, in a particular aspect, the invention provides for an in vitro method for distinguishing a hormone independent proliferative disorder or hormone-refractory proliferative disorder from a hormone-sensitive proliferative disorder in tissue and/or in at least one cell obtainable from tissue of the prostate from a subject. Such diagnostic method comprises contacting a DNA of a tissue or a DNA of a biological fluid with a reagent which detects the methylation status of the promoter region of the MSMB gene, wherein hypermethylation, as compared to the methylation status of the MSMB promoter region or upstream of the promoter region from a normal cell or compared to the methylation status of the MSMB promoter region or upstream of the promoter region from cells of tissue of a prostate with steroidal hormone-sensitive proliferative disorder, is indicative of the steroidal hormone-refractory proliferative disorder.
[0118]The test is particularly suitable to distinguish between hormone-refractory and hormone-sensitive and in particular for androgen-sensitive and androgen-refractory prostate proliferative disorders and to distinguish between benign prostate hyperplasia and prostate cancer.
[0119]In certain embodiments, provided is a method for distinguishing between androgen-sensitive and androgen-refractory prostate cancer by contacting a cellular component of a prostate tissue sample or another sample with a reagent which detects the methylation status of the MSMB promoter or upstream of the MSMB promoter region.
[0120]As aforementioned, methylation-sensitive restriction endonuclease can be utilized to identify a hypermethylated MSMB promoter or upstream region, for example.
[0121]Other approaches for detecting methylated CpG dinucleotide motifs use chemical reagents. In particular chemical reagents that selectively modify the methylated or non-methylated form of CpG dinucleotide motifs can be used in the methods of the invention. Such chemical reagents include bisulphite ions. Sodium bisulphite converts unmethylated cytosine to uracil but methylated cytosines remain unconverted. Analysis of the nucleic acid sequence after bisulfite conversion indicates if the original nucleic acid was all or not methylated.
[0122]Multiple techniques for analyzing the methylation status of CpG dinucleotide motifs in CpG islands are known in the art. They comprise without limitation sequencing, methylation-specific PCR (MS-PCR), McMS-PCR, MLPA, QAMA, MSRE-PCR, MethyLight, HeavyMethyl, ConLight-MSP, BS-MSP, COBRA, McCOBRA, MS-SNuPE, MS-SSCA, PyroMethA, MALDI-TOF, MassARRAY, ERMA, QBSUPT, MethylQuant, Quantitative PCR sequencing, oligonucleotide-based microarray systems, Pyrosequencing, and Meth-DOP-PCR. A review of techniques for the detection of the methylation state of a gene is given, for instance, in Oral Oncology 2006, Vol. 42, 5-13 and references cited therein.
[0123]A preferred technique for the detection and/or quantification of methylated DNA is the Methylation Specific PCR (MSP) technique. This technique can be used in end-point format, wherein the presence of methylated DNA is, for instance, detected by electrophoresis or by the use of dyes such as SYBR Green I or Ethidium Bromide that bind double-stranded DNA that accumulates during the amplification reaction. Alternatively, the method is based on the continuous optical monitoring of an amplification process and utilizes fluorescently labeled reagents. Their incorporation in a product can be quantified as the reaction processes and is used to calculate the copy number of that gene or sequence region in the sample. The quantification of the amplification product may require the use of controls to avoid false negativity/positivity of the reaction. Particularly suitable for the quantification of the amplification product are reference genes (e.g., beta-actin) whose methylation status is known, and/or DNA standards (e.g., methylated or unmethylated standards).
[0124]Accumulation of an amplification product can be monitored through the incorporation of labeled reagents. Some techniques use labeled primers; others rely upon the use of labeled probes to monitor the amplification product. Real-time quantitative methylation specific PCR techniques comprise the use of Amplifluor primers and/or Molecular Beacon probes and/or Fret probes and/or Scorpion primers and/or Taqman probes and/or oligonucleotide blockers (e.g., HeavyMethyl approach) and/or DzyNA primers. All these probes and primers have been described and their mode of action is well known in the art.
[0125]In certain embodiments, the methods of the invention use unmethylated specific primers indicated by SEQ ID NOS:15, 16, 18, 23, 24, 25, 27, 30, 31, 40, 41, 44, 45, 52, 53, 59, 60, 63, 64 and/or methylated specific primers indicated by SEQ ID NOS:13, 14, 17, 19, 20, 21, 22, 26, 28, 29, 38, 39, 42, 43, 50, 51, 57, 58, 61, 62.
[0126]Alternatively to PCR, other amplification methods such as NASBA, 3SR, TMA, LCR, selective amplification of target polynucleotide sequences (U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chain reaction (U.S. Pat. No. 5,437,975), arbitrarily primed polymerase chain reaction (WO 90/06995), invader technology, strand displacement technology, and nick displacement amplification (WO 2004/067726) may be used to amplify the appropriate nucleic acid.
[0127]In certain embodiments, bisulphite sequencing is utilized in order to determine the methylation status of the MSMB gene. Primers may be designed in both the sense and antisense orientation to direct sequencing across the relevant region of the MSMB gene. In one embodiment, bisulphite sequencing may be carried out by using at least one the following sequencing primers. SEQ ID NOS: 9, 10, 11, 12, 32, 33, 34, 35, 36, 37, 46, 47, 48, 49, 54, 55, 56, 65, 66, 67, 68, 69 and 70.
[0128]These amplification primers, amplification probes and sequencing primers form a further aspect of the invention.
[0129]Revealed is that a hypermethylated promoter for the regulatory regions of the human MSMB gene, CpG island in the promoter and upstream of the promoter, positively correlates with androgen-insensitivity in prostatic carcinogenesis. This invention provides a diagnostic tools or means to determine a prostate cancer and to distinguish between androgen sensitivity and androgen independency of such prostate cancer. Methylation changes are not only ideal for screening purposes, but also interesting targets for monitoring staging or grading of the cancer. Methods for identifying a prostate cell proliferative disorder in a subject, can comprise the steps of: [0130](a) obtaining a biological sample from the subject; [0131](b) determining the methylation state of CpG island upstream and/or downstream of the TSS region and/or in the promoter of the MSMB gene, for instance, the CpG islands about 3 kb upstream and about 2.2 kb downstream of the transcriptional start site in the MSMB gene in the subject's sample; and [0132](c) identifying hypermethylation of the region(s), wherein hypermethylation on CpG and/or non-CpG dinucleotides is identified as being different when to the same region(s) of the gene or associated regulatory region in a subject not having the prostate cellular proliferative disorder, wherein detection of hypermethylation is indicative for the stage or grade of the prostate cancer.
[0133]This unexpected finding allows diagnosis of hormone-independent cancers by a simple assay that detects the hypermethylated GCP islands in the promoter region or upstream of the promoter region directly by, for instance, restriction endonuclease analysis to select the proper treatment for subjects with a prostate cancer, depending on the fact of the prostate cancer is hormone-refractory or hormone-sensitive or depending on the stage or grade of prostate cancer as can be indicated by the hypermethylation status. This is more reliable than detecting levels of MSMB mRNA or MSMB gene products. The diagnostic methods will also allow indication of the proper treatment for hormone-refractory cancers or avoid giving subjects with a hormone-sensitive cancer an inadequate treatment or assure that they can be treated differently. For instance, patients by the diagnosis of the invention to have hypermethylation of a CpG island in the promoter region or upstream of the promoter region of the MSMB can be subjected to an antimitotic drug therapy methods of treatment or the treatment can now adequately be directed to replacing the hypermethylated CpG islands with a non-methylated islands, which, for instance, is possible by a treatment with a therapeutically sufficient dosage of a pharmaceutically acceptable DNA methylation inhibitor. A particular treatment selected based on the conclusion of the diagnosis method of the invention can also be a treatment to decrease the expression of the histone modifier gene, EZH2, or a treatment to decrease the activity of the EZH2 protein. Such treatments are available in the art. For instance, Chroma Therapeutics developed a series of compounds that inhibit specifically EZH2.
[0134]Methylation and hypermethylation of the non -CpG of the MSMB gene was found to be a biomarker to distinguish between benign prostate hyperplasia (BPH) and prostate cancer. Human samples 5 prostate cancer samples and 4 benign prostate hyperplasia (BPH) and one whole blood sample (Control) have been screened for methylations and sequence variations to map CpG methylation in the CpG4-5 region of the MSMB gene, to map non-CpG methylation in the CpG4-5 region of the MSMB gene and to map sequence variations in CpG4-5 region of the MSMB gene.
[0135]A weak hypermethylation on some CpG dinucleotides in the CpG4-5 island of the MSMB gene was found in the human prostate cancer samples in comparison to benign prostatic hyperplasia (Table 2). Indeed, after bisulphite sequencing, one out of eleven analyzed clones of benign prostate hyperplasia sample B3 was completely unmethylated, and five out of eighteen analyzed clones of benign prostate hyperplasia sample B4 was methylated less than 50%. In contrast, the average CpG methylation level for all clones from prostate cancer samples was about 90% (Table 2). No significant difference for CpG methylation in CpG4-5 island of the MSMB gene was found between human prostate cancer sample and human genomic DNA isolated from whole blood (Table 2).
[0136]A clear hypermethylation on some non-CpG dinucleotides in the CpG4-5 island of the MSMB gene was found in the human prostate cancer samples in comparison to benign prostatic hyperplasia (Table 3). Indeed, after bisulphite sequencing, four out of five prostate cancer samples contained clones with non-CpG methylation on position -2973 bp, -2958 bp, -2944 bp, -2885 bp towards the TSS of the MSMB gene, which was not observed in the clones from benign prostatic hyperplasia samples B1-5. Prostate Cancer sample C4 showed another non-CpG methylation that was located on position -3328 bp, -3194 bp, -3111 bp towards the TSS of the MSMB gene and which was also not observed in benign prostatic hyperplasia samples B1-5. CA-dinucleotide methylation at position -3223 bp and -3100 bp was found only in some clones from the benign prostate hyperplasia samples B4. CT-dinucleotide at position -2992 was methylated in some clones of both prostate cancer (C1, C5) and benign (B1, B2) samples. Importantly, non-CpG methylation was not at all detected in human genomic DNA isolated from whole Blood (sample H1) (Table 3).
[0137]All different sequence alterations which are detected in the CpG4-5 island of the MSMB gene in prostate cancer (C1-4), benign prostatic hyperplasia (B1-5) samples and whole blood (H1) are shown in Table 4.
[0138]In conclusion, analysis of DNA samples of five prostate cancer samples (C1-5), four benign prostatic hyperplasia (B1-4) and whole blood (H1) on differential non-CpG methylation and sequence alterations in CpG4-5 island of the MSMB gene resulted in total 10 new haplotypes (Table 5). Haplotype 0 corresponds to a reference sequence of the corresponding fragment of the MSMB gene without any alteration. Haplotypes 3, 4 and 6 are only found in prostate cancer and not in benign prostatic hyperplasia. Haplotype 3, present in C1, C2, C3 and C5, is characterized by non-CpG methylation at position -2973 bp, -2958 bp, -2944 bp and -2885 bp towards TSS of MSMB gene. Haplotype 4, present in sample C4, is characterized by CA methylation at position -3352 bp and sequence alteration at position -3166 bp, -2956 bp, -2782 bp towards TSS of MSMB gene. Haplotype 6, present in sample C4, is characterized by non-CpG methylation at position -3328 bp, -3194 bp and -3111 bp towards TSS of the MSMB gene. Haplotypes 7-10 were present only in benign prostate hyperplasia samples, while haplotypes 0, 1, 2 and 5 could be found both in cancer and benign prostate samples.
[0139]This unexpected finding allows to diagnose prostatic cells or tissues for prostate cancer and to distinguish between a condition of benign prostate hyperplasia and prostate cancer.
[0140]The unexpected findings of the invention now specifically allows to diagnose for androgen-independent prostate cancer (AIPC) by a simple assay that detects the hypermethylated promoter or upstream region of the promoter directly of the MSMB gene and to select the proper treatment for subjects with this AIPC or avoid that subjects with an androgen-sensitive cancer will receive an inadequate treatment or allow that a such subject will be treated differently than subjects with androgen-sensitive prostate cancer.
[0141]For instance, patients by the diagnosis of the invention found to have hypermethylation of a CpG island in the promoter region of the MSMB gene or in the regulatory regions surrounding the transcriptional start site of the MSMB gene can be subjected to an antimitotic drug therapy methods or the treatment can now focus on replacing the hypermethylated promoter with a non-methylated promoter.
[0142]Moreover, the unexpected findings of the invention now specifically allows to define of a patients has a prostate cancer or a benign prostate hyperplasia by a simple assay that detects methylated non CpG dinucleotides in the promoter or upstream region of the promoter or in the regulatory regions surrounding the transcriptional start site of the MSMB gene and to select the proper treatment for subjects with this benign prostate hyperplasia or prostate cancer or avoid that subjects with benign prostate hyperplasia will receive an inadequate anti-cancer treatment or allow that a such subject will be treated differently than subjects with prostate cancer.
[0143]Moreover, in the case of trimethylation of the histones, the treatment can focus on decreasing the expression of the EZH2 gene or a treatment to decrease the activity of the EZH2 protein. We have used quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) to examine whether these changes can also be detected in prostate-derived cell lines. The EZH2 transcript was indeed four- to 14-fold over-expressed in the androgen-refractory PC-3 and DU 145 cell lines, as compared to the EZH2 expression level in the androgen-sensitive PZ-HPV-7 and LNCaP cells (FIG. 1, Panel a). Furthermore, the PSP94 transcript was readily detected in the PZ-HPV-7 and LNCaP cells, but was at least three orders of magnitude less abundant in the PC-3 and DU 145 cells. Having confirmed an inverse relationship between the transcript levels of EZH2 and MSMB, we subsequently examined whether these changes are causally related and whether the MSMB gene is a target for H3K27 trimethylation. Using a chromatin immunoprecipitation procedure (ChIP) with anti-H3K27me3 antibodies, we found that the MSMB gene in PC-3 cells was heavily trimethylated on H3K27 in nucleosomes that were associated with the promoter region (primer set 2) and with flanking sequences (primer sets 1, 3 and 4) (FIG. 1, Panels b and c). In contrast, nucleosomes from a fragment of intron 3 of the MSMB gene (primer set 5) were much less trimethylated on H3K27. In these experiments MYT1 (myelin-transcription-factor 1), a well-established Polycomb target gene (Kirmizis et al., 2004), served as a positive control and glyceraldehyde-3 phosphate dehydrogenase (GAPDH) as a negative control.
[0144]Importantly, whereas the MSMB gene was heavily trimethylated on H3K27 in PC-3 cells, which hardly express PSP94, this gene was only mildly trimethylated on H3K27 in LNCaP cells, which express a lot of PSP94 (FIG. 1, Panels a and c). As EZH2 is the major histone methyltransferase known to trimethylate H3K27 in vivo, the above data suggested that the MSMB gene is a target for repression by EZH2. Consistent with this notion, ChIP experiments with anti-EZH2 antibodies showed an association of EZH2 with all the analyzed regions of the MSMB gene (FIG. 2, Panel a). Binding of EZH2 to the MSMB gene was as robust as its binding to the MYT1 gene, a well-known EZH2 target gene. Interestingly, neither MSMB nor MYT1 was abundantly trimethylated on H3K9. As H3K9 trimethylation also correlates with transcriptional repression but is EZH2-independent, these data attest to the specificity of the detected EZH2-H3K27me3 association. Little or no enrichment of GAPDH DNA was observed in the ChIP experiments with the different antibodies (FIG. 2, Panel a). To obtain more direct evidence for a role of EZH2 in the transcriptional repression of MSMB, we have subsequently examined the effect of the RNAi-mediated knockdown of EZH2 on the H3K27 trimethylation of the MSMB gene in PC-3 cells. As expected, less EZH2 was associated with the MSMB gene (FIG. 2, Panel c) following the knockdown of EZH2 (FIG. 2, Panel b). Within the time frame of the experiment (48 hours), the loss was only evident in intron I (primer sets 3 and 4) and was not detected in upstream (primer sets 1 and 2) or downstream (primer set 5) sequences. This is reminiscent of the local loss of the association of PRC2 component SUZ12 with the MYT1 gene following the knockdown of SUZ12 (Cao and Zhang, 2004). Importantly, the loss of the targeting of EZH2 to intron 1 was associated with a loss of H3K27 trimethylation in this region (FIG. 2. Panel d). We have also investigated whether a change in the level of EZH2 affects the expression of MSMB. In FIG. 2, Panel e, it is shown that the knockdown of the EZH2 transcript in PC-3 cells by about 70% was associated with a threefold increase in the expression of MSMB. The transcript level of a control housekeeping gene, PPP1R8, was not affected. As the knockdown of EZH2 only affected the targeting of EZH2 to intron 1 (FIG. 2, Panel c), this suggests that intron 1 harbors important regulatory elements of MSMB expression. Conversely, the overexpression of EZH2, fused to a Gal4-tag, in PZ-HPV-7 cells resulted in a 50% drop of the MSMB transcript level but was without effect on the transcript level of the housekeeping gene HPRT (FIG. 2, Panel f). Collectively, the above data demonstrate that MSMB is a canonical EZH2 target gene and that repression of MSMB is associated with trimethylation of H3K27. Polycomb target genes are often additionally silenced through histone deacetylation and DNA methylation of CpG islands (van der Vlag and Otte, 1999; Vire et al., 2006). This is explained by the ability of PcG proteins to bind histone deacetylases and to recruit DNA methyltransferases.
[0145]We used trichostatin A (TSA), a cell permeable inhibitor of histone deacetylases, to examine whether the MSMB gene is also controlled by histone (de)acetylation. The addition of TSA (50 ng/ml) to PC-3 cells for nine hours indeed resulted in a six-fold increase of the MSMB transcript level (FIG. 3, Panel a). As TSA did not affect the expression level of EZH2, these data strongly indicate that the MSMB gene is additionally silenced by histone deacetylation.
[0146]To examine our findings in more detail, we performed ChIP experiments with antibodies against acetylated Lys 9 of Histone H3 (H3K9ac). Three of the four examined regions of the MSMB gene were hypoacetylated in PC-3 cells, as compared to their acetylation status in LNCaPs (FIG. 3, Panel b). This fits nicely with the decreased expression of the MSMB gene in PC-3 cells (FIG. 1, Panel a) and is further evidence for a role of deacetylation in the repression of this gene in PC-3 cells. Finally, we have found that 50-azacytidine, an inhibitor of DNA methyltransferases, promotes the expression of the MSMB gene in PC-3 cells by about fivefold (FIG. 4, Panel a), indicating that DNA methylation also contributes to the repression of MSMB. In further agreement with this notion, we found that the MSMB gene harbors two CpG islands (FIG. 4, Panel b). DNA bisulfite sequencing revealed that these islands are indeed methylated, both in PC-3 and in LNCaP cells. Interestingly, the methylation of the CpG island in the promoter region was significantly more pronounced in PC-3 cells, as compared to its methylation in LNCaP cells, in agreement with the lesser expression of the MSMB gene in PC-3 cells. In addition, the methylation of this CpG island in PC-3 was decreased following the addition of 50-azacytidine, which is additional evidence that MSMB is controlled by DNA methylation.
[0147]Diagnosing the acetylation status of MSMB gene histones can be a further diagnostic tool to decide which of the prostate cancer patients should be treated by histone deacetylase inhibitors (HDAC inhibitors). Various histone deacetylase inhibitor are available in the art for and structurally defined for the man skilled in the art from several companies such as EntreMed, Merck & Co, Karus Therapeutics, Kalypsys (US20070123580 and US20070135438), Johnson & Johnson (JNJ 26481585, a second-generation, oral, pan-HDAC inhibitor with broad-spectrum preclincial antitumor activity), MethylGene/EnVivo (U.S. Pat. No. 7,288,567 B2, U.S. Pat. No. 6,946,441 B2, US20070155730 and U.S. Pat. No. 6,897,220 B2), ArQule, Sulfidris and Pharmacyclics (US20070281934), the compounds hereby incorporated by reference.
[0148]The HDAC inhibitors of Pharmacyclics comprise, for instance, a compound selected from among: 1-(3,4-dichloro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(2-methyl-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3,4,5-trimethoxy-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3-fluoro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3-methyl-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(benzyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3,5-dimethoxy-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(1-methyl-1-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(4-fluoro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(2-fluoro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(2-chloro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3-methoxy-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(naphth-2-ylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3-phenylpropyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(cyclohexylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-[1-(phenyl)-propen-3-yl]-1H-indole-6-carboxylic acid hydroxyamidel-[4-(trifluoromethoxy)-phenylmethyl]-1H-indole-6-carboxylic acid hydroxyamide; 1-(4-chloro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(benzo[2,1,3]oxadiazol-5-ylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(4-methyl-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3-fluoro-4-methoxy-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-[4-(difluoromethoxy)-phenylmethyl]-1H-indole-6-carboxylic acid hydroxyamide; 1-(4-methoxy-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(phenethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(3-chloro-phenylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-[N-(t-butoxycarbonyl)piperidin-4-ylmethyl]-1H-indole-6-carboxylic acid hydroxyamide; 1-(piperidin-4-ylmethyl)-1H-indole-6-carboxylic acid hydroxyamide; 1-(N-methylsulfonyl-3-aminobenzyl)-1H-indole-6-carboxylic acid hydroxyamide; 3-(Dimethylaminomethyl)-1-(4-methoxybenzyl)-1H-indole-6-carboxylic acid hydroxyamide; 3-(N-Morpholinomethyl)-1-(4-methoxybenzyl)-1H-indole-6-carboxylic acid hydroxyamide; 3-(N-Pyrrolidinomethyl)-1-(4-methoxybenzyl)-1H-indole-6-carboxylic acid hydroxyamide; 3-(N-Benzylaminomethyl)-1-(4-methoxybenzyl)-1H-indole-6-carboxylic acid hydroxyamide; and 3-(Ethyl)-1-(4-methoxybenzyl)-1H-indole-6-carboxylic acid hydroxyamide.
[0149]The HDAC inhibitors of EntreMed (EntreMed. Inc. EntreMed Completes Acquisition of Miikana Therapeutics, Media Release, 11 Jan. 2006. Available from: World Wide Web entremed.com have been developed by Miikana (US20050197336, US20050250784 and US20060199829) as series of mercaptoamide-based, non-hydroxamate inhibitors of HDACs. The structure of this series was designed to be as close as possible to that of vorinostat, but having a mercaptoamide replacing the hydroxamate moiety. Particular compounds are for instance the compounds of the group consisting of 1,3,4,9-tetrahydro-2H-b-carbolin-2-yl, 1,3,4,4a,9,9a-hexahydro-2H-b-carbolin-2-yl, 1,3,4,5-tetrahydro-2H-pyrido[4,3-b]indol-2-yl, 1,1a,3,4,4a,5-hexahydro-2H-pyrido[4,3-b]indol-2-yl, 1,4,5,6-tetrahydroazepino[4,5-b]indol-3(2H)-yl, 3,4-dihydro[1]benzothieno[2,3-c]pyridin-2(1H)-yl, 3,4-dihydro[1]benzofuro[2,3-c]pyridin-2(1H)-yl and 10-oxo-3,4,5,10-tetrahydrobenzo[b]-1,6-naphthyridin-2(1H)-yl. Other such inhibitors of HDACs are, for instance, -(2-naphthylsulfonyl)-4-(5-hydroxyaminocarbonylthiazol-2-yl)piperazine; 1-(2-naphthylsulfonyl)-4-(5-hydroxyamino-carbonylthiazol-2-yl)-1,4-diazep- ane; 1-(2-naphthylsulfonyl)-4-(4-hydroxyaminocarbonylthiazol-2-yl)piperazi- ne; 1-(2-naphthylsulfonyl)-4-[(5-(2-hydroxyaminocarbonylethen-1(Z)-yl-thia- zol-2-yl)piperazine; 4-(2-naphthylsulfonylamino)-1-[(5-(2-hydroxyaminocarbonyl-thiazol-2-yl)-p- iperadine; 2-[4-(3,4-dimethoxy-benzene sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-trifluoromethoxy-benzene sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-toluene-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-trifluoromethyl-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carbo- xylic acid hydroxyamide; 2-[4-(4-nitro-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-acetyl-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(thiophene-2-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(biphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(5-dimethylamino-naphthalene-1-sulfonyl)-piperazin-1-yl]-thiazole-5-- carboxylic acid hydroxyamide; 2-[4-(4-fluoro-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-(4-methyl-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxyamide; 2-(4-Benzyl-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxyamide; 2-(4-(2-hydroxyethyl)-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxyamide; 2-(4-(2-aminoethyl)-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxyamide; 2-(4-phenylethyl-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxyamide; 2-(4-acetyl-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxamide; 2-(4-benzoyl-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxamide; 2-(4-phenylacetyl-piperazin-1-yl)-thiazole-5-carboxylic acid hydroxamide; N-(2-naphthylsulfonyl)-N'-{2-[5-(N-hydroxycarboxamido)]thiazolyl}-piperaz- ine; and pharmaceutically acceptable salts, isomers, tautomers, and prodrugs thereof. 2-[4-(naphtha-2-yl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-trifluoromethoxy-benzene sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-toluene-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(biphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-trifluoromethyl-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carbo- xylic acid hydroxyamide; 2-[4-(3,4-dimethoxy-benzene sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(5-dimethylamino-naphthalene-1-sulfonyl)-piperazin-1-yl]-thiazole-5-- carboxylic acid hydroxyamide; 2-[4-(4-acetyl-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-nitro-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-fluoro-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-pyrolidinylbenzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(thiophene-2-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(N-methyl-2,3-dihydrobenzisoxazinylsulfonyl) piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-isopropylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(trans-2-phenylethanelsulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide; 2-[4-(2-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3,4-dichlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(N,N-dimethylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-methylsulfonylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxyl- ic acid hydroxyamide; 2-[4-(pyridine-3-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(2-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-[(dimethylamino)methyl]-1,1'-biphenylsulfonyl]piperazin-1-yl)}-- 1,3-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-n-propylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3,5-dimethylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-t-butylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(benzylsulfonyl)piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4'-N,N-dimethylcarboxamido-1,1'-biphenylsulfonyl) piperazin-1-yl]-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4'-methylsulfonylamino-1,1'-biphenylsulfonyl)piperazin-1-yl]-1,3-th- iazole-5-carboxylic acid hydroxyamide; 2-{4-[(4'-((dimethylamino)methyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1- ,3-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3,5-dimethylisoxazolesulfonyl)piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-({4-morpholino}-3-pyridylsulfonyl)-piperazin-1-yl]-thiazole-5-carbox- ylic acid hydroxyamide; 2-[4-(3'-(dimethylamino)-1,1'-biphenylsulfonyl)piperazin-1-yl]-1,3-thiazo- le-5-carboxylic acid hydroxyamide; 2-{4-[(3'-(pyrrolidin-1-ylmethyl 1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-dimethylaminophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide; 2-[4-(3,4-dimethylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4'-(morpholin-4-ylcarbonyl 1,1'-biphenylsulfonyl)piperazin-1-yl]-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[(2-((dimethylamino)-methyl)thien-3-yl]phenylsulfonyl}piperazin-1- -yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(4'-fluoro-1,1'-biphenyl-4-yl)sulfonyl]piperazin-1-yl}-1,3-thiazole- -5-carboxylic acid hydroxyamide; 2-[4-(3-chloro-2-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-[4-(3-chloro-4-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-[4-(4-methoxyphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[4-(pyridin-4-yl)phenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-carbo- xylic acid hydroxyamide; 2-[4-(3-fluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(2-fluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(2-methyl-5-fluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-[4-(2-trifluoromethylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-{4-[(3'-chloro-1,1'-biphenyl-4-yl)sulfonyl]piperazin-1-yl}-1,3-thiazole- -5-carboxylic acid hydroxyamide; 2-{4-[(2'-chloro-1,1'-biphenyl-4-yl)sulfonyl]piperazin-1-yl}-1,3-thiazole- -5-carboxylic acid hydroxyamide; 2-(4-{[4-(2-furyl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-carboxyl- ic acid hydroxyamide; 2-[4-(3,4-difluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-fluoro-2-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-[4-(3-fluoro-4-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-[4-(2-methyl-6-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-[4-(2,5-dimethyl-4-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-car- boxylic acid hydroxyamide; 2-[4-(2,1,3-benzothiadiazole-5-sulfonyl)-piperazin-1-yl]-thiazole-5-carbo- xylic acid hydroxyamide; 2-[4-(2-benzothiphenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(2,3-dihydrobenzofuransulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide; 2-[4-(3,4-benzodioxansulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3-biphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(2-phenoxypyridine-5-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(2-{2-methylthiopyrimidine-4-yl}-5-thiophenesulfonyl)-piperazin-1-yl- ]-thiazole-5-carboxylic acid hydroxyamide; 2-(4-[4-{(2-(pyrrolidin-1-ylmethyl)-thien-3-yl) phenylsulfonyl}]-piperazin-1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[(5-(pyrrolidin-1-ylmethyl)-thien-2-yl]phenylsulfonyl}-piperazin-- 1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-((4-methylpiperazin-1-yl)methyl)-1,1'-biphenylsulfonyl]-piperaz- in-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[(2-(dimethylamino)ethyl)(methyl)amino]methyl}-1,1'-biphenylsul- fonyl]piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3,5-difluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[(3-(pyrrolidin-1-ylmethyl)-thien-2-yl]phenylsulfonyl}-piperazin-- 1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-[isopropyl(methyl)amino]methyl]-1,1'-biphenylsulfonyl]-piperazi- n-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-[ethyl(methyl)amino]methyl]-1,1'-biphenylsulfonyl]-piperazin-1-- yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-fluoro-1,1'-biphenylsulfonyl]piperazin-1-yl})-1,3-thiazole-5-ca- rboxylic acid hydroxyamide; 2-(4-{[4-(1,3-benzodioxol-5-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazo- le-5-carboxylic acid hydroxyamide; 2-[4-(n-butylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(5-bromothien-2-yl)sulfonyl]piperazin-1-yl}-N-hydroxy-1,3-thiazole-- 5-carboxamide; 2-{4-[(4'-chloro-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-car- boxylic acid hydroxyamide; 2-{4-[(4'-methoxy-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-ca- rboxylic acid hydroxyamide; 2-{4-[(4'-(2,2-dimethylpropyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-- thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(4-thien-2-ylphenyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxy- lic acid hydroxamide; 2-(4-{[4-(1-(2,2-dimethylprop-oxycarbonyl)-1H-pyrrol-2-yl)phenyl]-sulfony- l}-piperazin-1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{[4-(1H-pyrrol-2-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-c- arboxylic acid hydroxyamide; 2-{4-[(3'-(piperidin-1-ylmethyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,- 3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-(4-methylpiperidin-1-ylmethyl 1,1'-biphenylsulfonyl]-piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-(hexahydroazepin-1-ylmethyl)-1,1'-biphenylsulfonyl]-piperazin-1- -yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-((diethylamino)methyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,- 3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(3'-((methyl(3-propenyl)amino)methyl)phenylsulfonyl]piperazin-1-yl}- -1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[(3-(pyrrolidin-1-ylmethyl)-2-furyl]phenylsulfonyl}piperazin-1-yl- )-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{5-[3-(pyrrolidin-1-ylmethyl)phenyl]thiophene-2-sulfonyl}piperazin-1- -yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-pyrzaol-1-yl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-{1-methylimidazol-4-yl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5-ca- rboxylic acid hydroxyamide; 2-[4-(4-methoxyphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-{3-trifluoromethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5-car- boxylic acid hydroxyamide; 2-[4-(4-{N,N-dimethylaminomethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiaz- ole-5-carboxylic acid hydroxyamide; 2-[4-(4-{N-morpholinomethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5- -carboxylic acid hydroxyamide; 2-[4-(4-{N-pyrrolidinylmethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole- -5-carboxylic acid hydroxyamide; 2-[4-(4-phenethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(4-ethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide;
2-[4-(3-hydroxymethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carbox- ylic acid hydroxyamide; 2-[4-(3-pyrrolidin-1-ylmethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-- 5-carboxylic acid hydroxyamide; 2-[4-(4-pyrimid-5-ylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide; 2-{4-[(4'-(acetamidophenyl)-phenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5- -carboxylic acid hydroxyamide; 2-[4-(3-pyridylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3-{N,N-dimethylaminomethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiaz- ole-5-carboxylic acid hydroxyamide; 2-[4-(3-methoxymethylbenzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxyl- ic acid hydroxyamide; 2-[4-(4-acetamido-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-[4-(3-cyanophenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[(2-chloro-5-methoxyphenyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-c- arboxylic acid hydroxyamide; 2-(4-{[3-(difluoromethoxy)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-- carboxylic acid hydroxyamide; 2-{4-[(4-methyl-3-nitrophenyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-car- boxylic acid hydroxyamide; 2-{4-[(2,5-dimethoxyphenyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-carbox- ylic acid hydroxyamide; 2-{4-[(2,5-dimethylphenyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxy- lic acid hydroxyamide; 2-(4-{[2-(pyrrolidin-1-ylmethyl-4-methylphenyl]sulfonyl}piperazin-1-yl)-1- ,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{[3-fluoro-4-(pyrrolidin-1-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-th- iazole-5-carboxylic acid hydroxyamide; 2-(4-{[4-(piperidin-1-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-c- arboxylic acid hydroxyamide; 2-(4-{[4-(morpholin-4-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-c- arboxylic acid hydroxyamide; 2-{4-[(trifluoromethyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[ethylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[N-acetylamino]methyl}-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,- 3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{methoxymethyl)}-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiaz- ole-5-carboxylic acid hydroxyamide; 2-{4-[4-(2-(pyrrolidin-1-ylmethyl)-thien-4-yl)phenylsulfonyl]piperazin-1-- yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[3-((dimethylamino)-methyl)-2-furyl]phenylsulfonyl}piperazin-1-yl- )-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[(4-(pyrrolidin-1-ylmethyl-2-furyl]phenylsulfonyl}piperazin-1-yl)- -1,3-thiazole-5-carboxylic acid hydroxyamide; 2-(4-{4-[(3-((dimethylamino)methyl)thien-2-yl]phenylsulfonyl}piperazin-1-- yl)-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[(2-hydroxyethyl)(methyl)amino]methyl}-1,1'-biphenylsulfonyl]pi- perazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[bis(2-hydroxyethyl)amino]methyl}-1,1'-biphenylsulfonyl]piperaz- in-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-(3,6-dihydropyridin-1(2H)-ylmethyl)-1,1'-biphenylsulfonyl]pipera- zin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[(butyl)(methyl)amino]methyl}-1,1'-biphenylsulfonyl]piperazin-1- -yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-((piperazin-1-yl)methyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1- ,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[(2-methoxyethyl)(methyl)amino]methyl}-1,1'-biphenylsulfonyl]pi- perazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[bis(3-propenyl))amino]methyl}-1,1'-biphenylsulfonyl]piperazin-- 1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide; 2-{4-[3'-{[methylamino]methyl}-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-- thiazole-5-carboxylic acid hydroxyamide and 2-{4-[2'-{pyrrolidin-1-ylmethyl}-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,- 3-thiazole-5-carboxylic acid hydroxyamide; and tautomers, isomers, prodrugs and pharmaceutically acceptable salts thereof. Yet other HDAC inhibitors are compounds selected from the group consisting of: 2-[4-(naphtha-2-yl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-trifluoromethoxy-benzene sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-toluene-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(biphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-trifluoromethyl-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carbo- xylic acid hydroxyamide 2-[4-(3,4-dimethoxy-benzene sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(5-dimethylamino-naphthalene-1-sulfonyl)-piperazin-1-yl]-thiazole-5-- carboxylic acid hydroxyamide 2-[4-(4-acetyl-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-nitro-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-fluoro-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-pyrrolidinylbenzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide 2-[4-(thiophene-2-benzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(N-methyl-2,3-dihydrobenzisoxazinylsulfonyl)-piperazin-1-yl]-thiazol- e-5-carboxylic acid hydroxyamide 2-[4-(4-isopropylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(trans-2-phenylethanelsulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide 2-[4-(2-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3,4-dichlorophenylsulfonyl)piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(N,N-dimethylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-methylsulfonylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxyl- ic acid hydroxyamide 2-[4-(pyridine-3-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(2-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-[(dimethylamino)methyl]-1,1'-biphenylsulfonyl]piperazin-1-yl}-1- ,3-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-n-propylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3,5-dimethylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-t-butylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(benzylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4'-N,N-dimethylcarboxamido-1,1'-biphenylsulfonyl)piperazin-1-yl]-1,- 3-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4'-methylsulfonylamino-1,1'-biphenylsulfonyl)piperazin-1-yl]-1,3-th- iazole-5-carboxylic acid hydroxyamide 2-{4-[(4'-((dimethylamino)methyl}1,1'-biphenylsulfonyl]piperazin-1-yl}-1,- 3-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3,5-dimethylisoxazolesulfonyl) piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-({4-morpholino}-3-pyridylsulfonyl)-piperazin-1-yl]-thiazole-5-carbox- ylic acid hydroxyamide 2-[4-(3'-(dimethylamino)-1,1'-biphenylsulfonyl)piperazin-1-yl]-1,3-thiazo- le-5-carboxylic acid hydroxyamide 2-{4-[(3'-(pyrrolidin-1-ylmethyl 1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-dimethylaminophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide 2-[4-(3,4-dimethylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4'-(morpholin-4-ylcarbonyl)-1,1'-biphenylsulfonyl)piperazin-1-yl]-1- ,3-thiazole-5-carboxylic acid hydroxyamide 2-(4-{4-[(2-((dimethylamino)-methyl)thien-3-yl]phenylsulfonyl}piperazin-1- -yl)-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(4'-fluoro-1,1'-biphenyl-4-yl)sulfonyl]piperazin-1-yl}-1,3-thiazole- -5-carboxylic acid hydroxyamide 2-[4-(3-chloro-2-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-[4-(3-chloro-4-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-[4-(4-methoxyphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-{4-[4-(pyridin-4-yl)phenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-carbo- xylic acid hydroxyamide 2-[4-(3-fluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(2-fluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(2-methyl-5-fluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-[4-(2-trifluoromethylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-{4-[(3'-chloro-1,1'-biphenyl-4-yl)sulfonyl]piperazin-1-yl}-1,3-thiazole- -5-carboxylic acid hydroxyamide 2-{4-[(2'-chloro-1,1'-biphenyl-4-yl)sulfonyl]piperazin-1-yl}-1,3-thiazole- -5-carboxylic acid hydroxyamide 2-(4-{[4-(2-furyl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-carboxyl- ic acid hydroxyamide 2-[4-(3,4-difluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-fluoro-2-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-[4-(3-fluoro-4-methylphenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-[4-(2-methyl-6-chlorophenylsulfonyl)piperazin-1-yl]-thiazole-5-carboxyl- ic acid hydroxyamide 2-[4-(2,5-dimethyl-4-chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-car- boxylic acid hydroxyamide 2-[4-(2,1,3-benzothiadiazole-5-sulfonyl)-piperazin-1-yl]-thiazole-5-carbo- xylic acid hydroxyamide 2-[4-(2-benzothiphenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(2,3-dihydrobenzofuransulfonyl)-piperazin-1-yl]-thiazole-5-carboxyli- c acid hydroxyamide 2-[4-(3,4-benzodioxansulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3-biphenylsulfonyl)piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(2-phenoxypyridine-5-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(2-{2-methylthiopyrimidine-4-yl}-5-thiophenesulfonyl)-piperazin-1-yl- ]-thiazole-5-carboxylic acid hydroxyamide 2-(4-[4-{(2-(pyrrolidin-1-ylmethyl)-thien-3-yl)phenylsulfonyl}]-piperazin- -1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide 2-(4-{4-[(5-(pyrrolidin-1-ylmethyl)-thien-2-yl]phenylsulfonyl}-piperazin-- 1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-((4-methylpiperazin-1-yl)methyl)-1,1'-biphenylsulfonyl]-piperaz- in-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[3'-{[(2-(dimethylamino) ethyl)(methyl)amino]methyl}-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thi- azole-5-carboxylic acid hydroxyamide 2-[4-(3,5-difluorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-(4-{4-[(3-(pyrrolidin-1-ylmethyl)-thien-2-yl]phenylsulfonyl}-piperazin-- 1-yl)-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-[isopropyl(methyl)amino]methyl]-1,1'-biphenylsulfonyl]-piperazi- n-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-[ethyl(methyl)amino]methyl]-1,1'-biphenylsulfonyl]-piperazin-1-- yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-fluoro-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-car- boxylic acid hydroxyamide 2-(4-{[4-(1,3-benzodioxol-5-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazo- le-5-carboxylic acid hydroxyamide 2-[4-(n-butylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(chlorophenylsulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(5-bromothien-2-yl)sulfonyl]piperazin-1-yl}-N-hydroxy-1,3-thiazole-- 5-carboxamide 2-{4-[(4'-chloro-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-car- boxylic acid hydroxyamide 2-{4-[(4'-methoxy-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-ca- rboxylic acid hydroxyamide 2-{4-[(4'-(2,2-dimethylpropyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-- thiazole-5-carboxylic acid hydroxyamide 2-{4-[(4-thien-2-ylphenyl)sulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxy- lic acid hydroxamide 2-(4-{[4-(1-(2,2-dimethylprop-oxycarbonyl 1H-pyrrol-2-yl)phenyl]-sulfonyl}-piperazin-1-yl)-1,3-thiazole-5-carboxyli- c acid hydroxyamide 2-(4-{[4-(1H-pyrrol-2-yl)phenyl]sulfonyl}piperazin-1-yl)-1,3-thiazole-5-c- arboxylic acid hydroxyamide 2-{4-[(3'-(piperidin-1-ylmethyl)-1,1'-biphenylsulfonyl]piperazin-1-yl}-1,- 3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-(4-methylpiperidin-1-ylmethyl)-1,1'-biphenylsulfonyl]-piperazin- -1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-(hexahydroazepin-1-ylmethyl)-1,1'-biphenylsulfonyl]-piperazin-1- -yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-((diethylamino)methyl} 1,1'-biphenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5-carboxylic acid hydroxyamide 2-{4-[(3'-((methyl(3-propenyl)amino)methyl)phenylsulfonyl]piperazin-1-yl}- -1,3-thiazole-5-carboxylic acid hydroxyamide 2-(4-{4-[(3-(pyrrolidine-1-ylmethyl)-2-furyl]phenylsulfonyl}piperazin-1-y- l)-1,3-thiazole-5-carboxylic acid hydroxyamide 2-(4-{5-[3-(pyrrolidin-1-ylmethyl)phenyl]thiophene-2-sulfonyl}piperazin-1- -yl)-1,3-thiazole-5-carboxylic acid hydroxyamide
2-[4-(4-pyrzaol-1-yl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-{1-methylimidazol-4-yl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5-ca- rboxylic acid hydroxyamide 2-[4-(4-methoxyphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-{3-trifluoromethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5-car- boxylic acid hydroxyamide 2-[4-(4-{N,N-dimethylaminomethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiaz- ole-5-carboxylic acid hydroxyamide 2-[4-(4-{N-morpholinomethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5- -carboxylic acid hydroxyamide 2-[4-(4-{N-pyrrolidinylmethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole- -5-carboxylic acid hydroxyamide 2-[4-(4-phenethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(4-ethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3-hydroxymethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carbox- ylic acid hydroxyamide 2-[4-(3-pyrrolidin-1-ylmethylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-- 5-carboxylic acid hydroxyamide 2-[4-(4-pyrimid-5-ylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxy- lic acid hydroxyamide 2-{4-[(4'-(acetamidophenyl)-phenylsulfonyl]piperazin-1-yl}-1,3-thiazole-5- -carboxylic acid hydroxyamide 2-[4-(3-pyridylphenyl-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3-{N,N-dimethylaminomethylphenyl}-4-sulfonyl)-piperazin-1-yl]-thiaz- ole-5-carboxylic acid hydroxyamide 2-[4-(3-methoxymethylbenzenesulfonyl)-piperazin-1-yl]-thiazole-5-carboxyl- ic acid hydroxyamide 2-[4-(4-acetamido-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide 2-[4-(3-cyanophenyl}-4-sulfonyl)-piperazin-1-yl]-thiazole-5-carboxylic acid hydroxyamide or tautomers, isomers, prodrugs and pharmaceutically acceptable salts thereof.
EXAMPLES AND DRAWING DESCRIPTION
Example 1
DNA Methylation Analysis
[0150]DNA methylation is an epigenetic event that affects cell function by altering gene expression and refers to the covalent addition of a methyl group, catalyzed by DNA methyltransferase (DNMT), to the 5-carbon of cytosine in a CpG dinucleotide. Methods for DNA methylation analysis can be divided roughly into two types: global and gene-specific methylation analysis. For global methylation analysis, there are methods which measure the overall level of methyl cytosines in genome such as chromatographic methods and methyl accepting capacity assay. For gene-specific methylation analysis, a large number of techniques have been developed. Most early studies used methylation-sensitive restriction enzymes to digest DNA followed by Southern detection or PCR amplification. Recently, bisulfite reaction-based methods have become very popular such as methylation specific PCR (MSP), bisulfite genomic sequencing PCR. Additionally, in order to identify unknown methylation hot-spots or methylated CpG islands in the genome, several of genome-wide screen methods have been invented such as Restriction Landmark Genomic Scanning for Methylation (RLGS-M), and CpG island microarray. For the various aspects of this technology is available in the art. The bisulfite modification (conversion) of DNA is obtainable using sodium bisulfite to convert unmethylated cytosines to uracils and subsequently detecting methylated cytosines using methylation specific PCR (MSP) technique or bisulfite genomic sequencing after PCR amplification with or without cloning. Protocols or procedures for handling such nanogram quantities of DNA have been published in technical manuals and journals (H. Hayatsu et al., Biochemistry 1970, 9:2858; K. D. Tremblay, 1998 Technical Tips Online; S. J. Clark et al., Nucleic Acids Res. 1994, 22:1827; M. Frommer et al., PNAS 1992, 89:1827; E. J. Oakeley, Pharmacology & Therapeutics 1999, 84:389; M. F. Fraga and M. Esteller, Biotechniques 2002, 33:632; L. C. Li and R. Dahiya, Bioinformatics 2002, 18(11):1427; and UCLA Fan Lab Bisulfite Treatment, by Shaun Fouse, online or via Dr. Guoping Fan, Department of Human Genetics, David Geffen School of Medicine, Gonda BLDG Rm. 6554, P.O. Box 957088, University of California Los Angeles, Los Angeles, Calif. 90095-7088). The analysis of DNA methylation can be carried out by bisulphite sequencing. This method allows precise analysis of methylation in a certain region by converting all nonmethylated cytosines into tymines, while methylated cytosines remain unchanged. This method requires small amount of genomic DNA and therefore seems to be very useful for the analysis of clinical samples, where the material amount is limited. Such method can be optimizes the method using genomic DNA from a cell line and then apply it to valuable samples. Primers have to be developed for bisulphite converted DNA. One can generate a model of bisulphite treated DNA by substituting all cytosines which are not in CG context into tymines. And then design the primers in the way that they don not contain any CG. If this is impossible, one can use C/T at the place of C in CG context. Usually primer selection is the most critical in bisulphite-based methylation analysis, since the complexity of DNA is reduced. Therefore the skilled man can select two to three pairs of primers, check them on bisulphite modified DNA, and use the most specific ones. Protocols and procedures of such analysis of DNA methylation by bisulphite sequences are available for the man skilled in the art (A. Kaneda et al., Cancer Lett. 2004, 212:203-210; and A. Kaneda et al., Cancer Sci. 2004, 95:58-64).
Example 2
Quantitative Methyl-Specific PCR (qMSP)
[0151]Quantitative Methyl-Specific PCR (qMSP) (J. G. Herman et al., Proc. Natl. Acad. Sci. USA Vol. 93, pp. 9821-9826, September 1996) is the ideal technology for early detection of PrCa. The detection of DNA methylation is based on bisulphite treatment of DNA, which reproducibly converts unmethylated cytosine into uracil. In this assay, the methylated cytosines remain unchanged. The bisulphite conversion is combined with a PCR-based approach known as Methylation-Specific PCR (MSP). This technique has been described enabling in U.S. Pat. Nos. 5,786,146, 6,017,704, 6,265,171, and 6,200,756 and is hereby incorporated by reference. It has major advantages over other PrCa screening methods:
[0152]The qMSP technology is extremely sensitive and can detect one to ten tumor cells among thousands of healthy cells.
[0153]The qMSP technology is highly reproducible, quick and easy to perform.
[0154]It is a low-cost assay only requiring standard qPCR equipment.
[0155]Since DNA is very stable (more stable than proteins), the qMSP test can be performed on many sample types, including DNA isolated from paraffin embedded, formalin fixed prostatectomy samples as well as body fluids such as blood plasma, urine or prostatic secretions (M. L. Gonzalgo et al. (2004), Urology 63:414-418).
Example 3
Predicted CpG Islands from the MSMB Gene
[0156]A detailed bioinformatical analysis of 15 kbp region upstream and downstream of the transcriptional start site of the MSMB gene resulted in the prediction of 10 CpG islands. All CpG islands, except for CpG5 and 7, are predicted by the program Newcpgreport, with the following parameters: window=50, window shift=1, Island size>200, GC %>0.2 and O/E (observed/predicted>0.2. CpG5 and 7 island are predicted by the program Methprimer with the following parameters: Island size>100, GC %>0.5 and O/E>0.6 A schematic representation and localization of the predicted CpG islands has been displayed in FIG. 6 and Table 1.
Example 4
Non-CpG Methylation in CpG4-5 Islands of the MSMB Gene
Location, See Table 1
[0157]Genomic DNA has been isolated from five prostate cancer samples, indicated as C1-5, and four Benign Prostatic Hyperplasia, indicated as B1-4 with GenElute Mammalian Genomic DNA Miniprep kit of Sigma. Human genomic DNA from whole blood, indicated as H, was purchased from Clonetech (cat no. 636401). Total genomic DNA of all samples, as well as the human genomic DNA from the whole blood, was bisulphite treated converting unmethylated cytosines to uracil. Methylated cytosines remained conserved. Bisulphite treatment was performed using the Zymo gold kit of Zymo Research (USA) according to the protocol of the manufacturer. After bisulphite conversion 10 ng of each DNA sample was used in a subsequent PCR to amplify the CpG islands 4 and 5 as one 799 bp fragment (sense primer is SEQ ID NO:67 and antisense primer is SEQ ID NO:68). Each reaction contained the following: 400 μM dNTPs, 10 pmol each primer, 2 U JumpStart® Taq DNA Polymerase (Sigma), 10 ng DNA (bisulphite treated). Denaturation at 95° C. for 4 minutes was followed by five cycles (95° C. for 30 seconds, annealing at 53° C. for 90 seconds, elongation at 68° C. for 120 seconds) and then by 30 cycles (95° C. for 30 seconds, annealing at 53° C. for 90 seconds, elongation at 68° C. for 90 seconds). A final elongation at 68° C. was carried out for five minutes. PCR products were subcloned in the pGem-T vector (Promega) according the manufacturer's protocol. The plasmids were sequenced using ABI Big Dye Terminator Cycle Sequencing Kit (Applied Biosystems). We determined the methylation status of all CG dinucleotides, known as CpG methylation, and of some CA, CT, CC dinucleotides, known as non-CpG methylation.
[0158]Table 2. Map of the methylation state of all CG dinucleotides present in CpG 4-5 island of the MSMB gene. The CpG 4-5 island is located at -3533 nt upstream to -2734 nt upstream of the transcriptional start site (TSS) of the MSMB gene. The TSS is located at position 51219559 on the forward strand of chromosome 10 (SEQ ID NO:5). The numbers in the first row represent the location of all CG dinucleotides towards the TSS of the MSMB gene (SEQ ID NO:5). Methylated CG dinucleotides are denoted by C and unmethylated CGs by T. Deleted CG-dinucleotides in some clones are denoted by DEL. A CG-dinucleotide in a position -3234 towards the TSS that is mutated in some DNA samples (CG/GG mutation), is denoted by GG. Non-available data for the methylation status of some CG-dinucleotides in some clones are denoted by hyphen. In the first column, the name of the analyzed biological sample followed by the number of the analyzed clone is indicated. C1-5, prostate cancer samples; B1-4, Benign Prostatic Hyperplasia; H, human genomic DNA.
[0159]Table 3. Map of the non-CG dinucleotides which are present in CpG4-5 island of the MSMB gene and were methylated in at least one of the analyzed samples. The CpG4-5 island is located at -3533 nt upstream to -2734 nt upstream of the transcriptional start site (TSS) of the MSMB gene. The TSS is located at position 51219559 on the forward strand of chromosome 10 (SEQ ID NO:5). The numbers in first row are the location of the non-CG dinucleotides towards the TSS of the MSMB gene (SEQ ID NO:5). Methylated non-CG dinucleotides are denoted by C, followed by the next nucleotide (A or T) and indicated by grey boxes. Unmethylated non-CG dinucleotides are denoted by T followed by the next nucleotide (A or T). In the first column, you find the name of the analyzed biological sample followed by the number of the analyzed clone. C1-5, prostate cancer sample; B1-4, Benign Prostatic Hyperplasia; H, human genomic DNA.
[0160]Table 4. Map of the sequence alterations unrelated to DNA methylation present in CpG4-5 island of the MSMB gene and detected in at least one analyzed sample. The CpG4-5 island is located at -3533 nt upstream to -2734 nt upstream of the transcriptional start site (TSS) of the MSMB gene. The TSS is located at position 51219559 on the forward strand of chromosome 10 (SEQ ID NO:5). The numbers in the first row refer to the location of the nucleotide towards the TSS of the MSMB gene (SEQ ID NO:5). Letter in the table indicates the nucleotide (A, T, G or C) detected in the indicate clone; all sequence alterations are marked by grey color. Deleted AGT-trinucleotide at position -2969 towards TSS of the MSMB gene in some clones is denoted by DEL. In the first column, the name of the analyzed biological sample followed by the number of the analyzed clone is indicated. C1-5, prostate cancer sample; B1-4, Benign Prostatic Hyperplasia; H, human genomic DNA.
[0161]Table 5. Summary of all detected haplotypes (differential non-CpG methylation plus sequence alterations unrelated to DNA methylation) of CpG4-5 island of the MSMB gene in all analyzed DNA samples. The CpG4-5 island is located at -3533 nt upstream to -2734 nt upstream of the transcriptional start site (TSS) of the MSMB gene. The TSS is located at position 51219559 on the forward strand of chromosome 10 (SEQ ID NO:5). The 11 haplotypes (numbered in the first column) are generated based on data of differential non-CpG methylation (Table 3) and sequence alterations from Table 4. The numbers in first row are the location of the nucleotide towards the TSS of the MSMB gene (SEQ ID NO:5). In the first row the non-CpG dinucleotides are denoted in grey boxes and the sequence alterations unrelated to DNA methylation in dotted boxes. A star in the two last columns indicates the presence of the haplotype in at least one cancer and/or benign samples; a hyphen, correspondingly, the absence of the haplotype in all analyzed cancer and/or benign samples. A star in the two last rows indicates the presence of the definite nucleotide modification in at least one cancer and/or benign samples; a hyphen, correspondingly, the absence of the definite nucleotide modification in all analyzed cancer and/or benign samples.
[0162]It will be apparent to those skilled in the art that various modifications and variations can be made in defining the methylation levels of the promoter region of the MSMB gene of the invention and in construction of the system and method without departing from the scope or spirit of the invention. Examples of such modifications have been previously provided. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
TABLE-US-00001 TABLE 1 Location of the predicted CpG islands towards the transcriptional start site of the MSMB gene. location towards TSS of the MSMB CpG island gene (SEQ ID NO: 5) CpG1 -13877 to -13583 CpG2 -10528 to -10254 CpG3 -3920 to -3673 CpG4 -3471 to -3141 CpG5* -3128 to -2817 CpG4-5** -3533 to -2734 CpG6 -1671 to 1996 CpG7*** 2180 to 2390 CpG8 5236 to 5616 CpG9 7670 to 8030 CpG10 11432 to 11754 *corresponds to CpG1 island in FIG. 4 **CpG4 island and CpG5 island are analyzed together by one PCR reaction and is indicated as CpG4-5 island ***corresponds to CpG2 island in FIG. 4
TABLE-US-00002 TABLE 2 CpG-methylation in CpG-island 4-5 of the MSMB gene -3234 -3408 -3383 -3376 -3345 -3312 -3308 -3281 -3250 -3244 CG-44 -3174 Clone CG-53 CG-52 CG-51 CG-50 CG-49 CG-48 CG-47 CG-46 CG-45 C-G GC-41 C1/24 C C C C C C C C C GG C C1/7 C C C C C C C C C GG C C1/1 C C C C C C C C C GG C C1/3 C C C C C C C C C GG C C1/8 C C C C C C C C C GG C C1/5 C C C C C C C C C GG C C1/11 C C C C C C C C C GG C C1/10 C C C C C C C C C CG C C1/2 C C C C C C C C C CG C C1/12 C C C C C C C C C CG C C1/15 C C C C C C C C C CG C C1/21 C C C C C C C C C CG C C1/18 C C C C C C C C C CG C C1/22 C C C C C C C C C CG C C1/4 C C C C C C C C C CG C C1/9 C C C C C C C C C CG C C1/16 C C C C C C C C C CG C C1/13 C C C C C C C C C CG C C1/23 C C C C C C C C C GC C C1/14 C C C C C C C C C CG C C1/20 C C C T C C C C C CG C C1/17 C C C T C C C C C CG C C1/6 C C C T C C C C C CG C C2/8 -- -- -- -- -- -- C C C CG C C2/4 C C C C C C C C C CG C C2/11 C C C C C C C C C CG C C2/19 C C C C C C C C C CG C C2/12 C C C C C C C C C CG C C2/2 C C C C C C C C C CG C C2/6 C C C C C C C C C CG C C2/10 C C C C C C C C C CG C C2/3 C C C C C C C C C CG C C2/13 C C C C C C C C C CG C C2/14 C C C C C C C C C CG C C2/16 C C C C C C C C C CG C C2/15 C C C C C C C C C CG C C2/17 C C C C C C C C C CG C C2/9 C C C C C C C C C CG C C2/24 T C C C C C C C C CG C C2/5 C C C C C C C C C CG C C2/7 C C C C C C C C C CG C C2/18 T C C C C C C C C CG C C2/22 T C C C C C C C C CG C C2/23 C C C C C C C C C CG C C3/22 C C C C C C C C C CG C C3/19 C C C C C C C C C CG C C3/11 C C C C C C C C C CG C C3/2 -- C C C C C C C C CG C C3/7 C C C C C C C C C CG C C3/10 C C C C C C C C C CG C C3/12 C C C C C C C C C CG C C3/9 C C C C C C C C C CG C C3/1 C C C C C C C C C CG C C3/13 C C C C C C C C C CG C C3/18 C C C C C C C C C CG C C3/16 C C C C C C C C C CG C C3/14 C C C C C C C C C CG C C3/23 C C C C C C C C C CG C C3/15 C C C C C C C C T CG C C3/20 C C C C C C C C C CG C C3/17 C C C C C C C C C CG C C3/24 C C C C C C C C C CG C C3/3 C C C C C C C C C CG C C3/8 C C C C C C C C C CG C C3/6 T C C C C C C C C CG C C4/11 C C C C C C C C C CG C C4/10 C C C C C C C C C CG C C4/4 C C C C C C C C C CG C C4/3 C C C C C C C C C CG C C4/1 C C C C C C C C C CG C C4/17 C C C C C C C C C CG C C4/14 C C C C C C C C C CG C C4/20 C C C C C C C C C CG C C4/18 C C C C C C C C C CG C C4/2 C C C C C C C C C CG C C4/12 C C C C C C C C C CG C C4/5 C C C C C C C C C CG C C4/8 C C C C C C C C C CG C C4/9 C C C C C C C C C CG C C4/21 C C C C C C C C C CG C C4/22 C C C C C C C C C CG C C4/13 C C C C C C C C C CG C C4/23 C C C C C C C C C CG C C4/15 C C C C C C C C C CG C C4/24 C C C T C C C C C CG C C4/16 C C C C C C C C C CG C C4/19 C C C C C C C T C CG C C5/7 C C C C C C C C C GG C C5/5 C C C C C C C C C GG C C5/1 C C C C C C C C C GG C C5/6 C C C C C C C C C GG C C5/11 C C C C C C C C C GG C C5/8 C C C C C C C C C GG C C5/10 C C C C C C C C C CG C C5/9 C C C C C C C C C CG C C5/2 C C C C C C C C C CG C C5/3 C C C C C C C C C CG C C5/4 -- -- -- C -- -- C T -- CG C C5/12 C C C C C C C C C CG C B1/2 T C C C C C C C C CG C B1/4 T C C C C C C C C CG C B1/15 T C C C C C C C C CG C B1/8 T C C C C C C C C CG C B1/14 T C C C C C C C C CG C B1/9 T C C C C C C C C CG C B1/13 C C C C C C C C C GG C B1/11 C C C C C C C C C GG C B1/12 C C C C C C C C C GG C B1/16 C C C C C C C C C GG C B1/6 C C C C C C C C C GG C B1/1 C C C C C C C C C GG C B1/3 C C C C C C C C C GG C B1/5 C C C C C C C C C GG C B1/7 C C C C C C C C C GG C B2/10 C C C C C C C C C GG C B2/5 C C C C C C C C C GG C B2/11 C C C C C C C C C GG C B2/7 C C C C C C C C C GG C B2/1 C C C C C C C C C GG C B2/2 C C C C C C C C C GG C B2/12 C C C C C C C C C GG C B2/3 C C C C C C C C C GG C B2/13 C C C C C C C C C CG T B2/4 C C C C C C C C C CG T B2/6 T C C C C C C C T CG C B2/9 C C C C C C C C C TG C B3/9 T C C C C C C C C CG C B3/8 C C C C C C C C C CG C B3/4 -- -- -- -- -- -- -- -- -- -- -- B3/10 T T T T T T T T T TG T B3/5 C C C T C C C C C CG C B3/6 C C C T C C C C C CG C B3/3 C C C T C C C C C CG C B3/1 C C C T C C C T C CG C B3/11 C C C T C C C C C CG C B3/2 C C C T C C C C C CG C B3/12 C C -- T C C C -- C CG C B4/10 -- -- T C C T T T T CG C B4/7 T C T C C T T T T CG C B4/2 T C T C C T T T T CG C B4/3 T C T C C T T T T CG C B4/17 T C T C C T T T T CG C B4/14 T C C C C T C C T TG C B4/18 T C C C C T C C T TG C B4/11 C C C C C C C C C CG C B4/13 C C C C C C C C C CG C B4/12 C C C C C C C C C CG C B4/1 C C C C C C C C C CG C B4/16 C C C C C C C C C CG C B4/4 C T C C C C C C C CG C B4/6 C T C C C C C C C CG C B4/5 C T C C C C C C C CG C B4/20 C T C C C C C C C CG C B4/21 C T C C C C C C C CG C B4/9 C C C C C C C C C CG C H1/1 C C C C C C C C C CG C H1/9 C C T C C C C C C CG C H1/5 C C C C C C C T C CG C H1/10 C C C C C C C C C CG C H1/2 C C C C C C C C C CG C H1/11 C C C T C C C C C CG C H1/8 T T C C C C C C C CG C H1/7 C C C C C C C C C CG C H1/12 C C C C C C C C C CG C H1/6 C C C C C C C C C CG C H1/4 C C C C C C C C C CG C H2/1 C C C C C C C C C CG C H2/12 C C C C C C C C C CG C H2/2 C C C C C C C C C CG C H2/5 C C C C C C C C C CG C H2/3 C C C T C C C C C CG C H2/8 C C C C C C C C C CG C H2/4 C C C C C C T C T CG C H2/10 C T C C C C C C C TG C H2/11 T C C C C C C C C CG C H2/6 C C C C C C C C C CG C H2/7 C C C C C C C C C CG C H2/9 C C T C C C C T T CG C -3097 -3065 -3057 -3035 -3033 -3026 3010 -3003 -2995 -2971 Clone CG-40 CG-39 CG-38 CG-37 CG-36 CG-35 CG-34C-A CG-33 CG-32 CG-30 C1/24 C C C C C C GC C C C C1/7 C C C C C C CG C C C C1/1 C C C C C C CG C C C C1/3 C C C C C C CG C C C C1/8 C C C C C C CG C C C C1/5 C C C C C C CG C C C C1/11 C C C C C C CG C C C C1/10 C C C C C C CG C C C C1/2 C C C C C C CG C C C C1/12 C C C C C C CG C C C C1/15 C C C C C C CG C C C C1/21 C C C C C C CG C C C C1/18 C C C C C C CG C C C C1/22 C C C C C C CG C C C C1/4 C C C C C C CG C C C C1/9 C C C C C C CG C C C C1/16 C C C C C C CG C C C C1/13 C C C C C C CG C C C C1/23 C C C C C C CG C C C C1/14 C C C C C C CG C C C C1/20 C C C C C C CG C C C C1/17 C C C C C C CG C C C C1/6 C C C C C C CG C C C C2/8 C C C C C C CG C C C C2/4 C C C C C C -- C C C C2/11 C C C C C C CG C C C C2/19 C C C C C C CG C C C C2/12 C C C C C C CG C C C C2/2 C C C C C C CG C C C C2/6 C C C C C C CG C C C C2/10 C C C C C C CG C C C C2/3 C C C C C C CG C C C C2/13 C C C C C C CG C C C C2/14 C C C C C C CG C C C C2/16 C C C C C C CG C C C C2/15 C C C C C C CG C C C C2/17 C C C C C C CG C C C C2/9 C C C C C C CG C C C C2/24 C C C C C C CG C C C C2/5 C C C C C C CG C C C C2/7 C C C C C C CG C C C C2/18 C C C C C C CG C C C C2/22 C C C C C C CG C C C C2/23 C C C C C C CG C C C C3/22 C C C C C C C C C C C3/19 C C C C C C C C C C C3/11 C C C C C C C C C C C3/2 C C C C C C C C C C C3/7 C C C C C C C C C C C3/10 C C C C C C C C C C C3/12 C C C C C C C C C C C3/9 C C C C C C C C C C C3/1 C C C C C C C C C C C3/13 C C C c C C C C C C C3/18 C C C T C C C C C C C3/16 C C C C C C C C C C C3/14 C C C C C C C C C C C3/23 C C C C C C C C C C C3/15 C C C C C C C C C C
C3/20 C C C C C C C C C C C3/17 C C C C C C C C C C C3/24 C C C C C C C C C C C3/3 C C C C C C C C C C C3/8 C C C C C C C C C C C3/6 C C C C C C C C C C C4/11 C C C C C C C C C C C4/10 C C C C C C C C C C C4/4 C C C C C C C C C C C4/3 C C C C C C C C C C C4/1 C C C C C C C C C C C4/17 C C C C C C C C C C C4/14 C C C C C C C C C C C4/20 C C C C C C C C C C C4/18 C C C C C C C C C C C4/2 C C C C C C C C C C C4/12 C C C C C C C C C C C4/5 C C C C C C C C C C C4/8 C C C C C C C C C C C4/9 C C C C C C C C C C C4/21 C C C C C C C C C C C4/22 C C C C C C C C C C C4/13 C C C C C C C C C C C4/23 C C C C C C C C C C C4/15 C C C C C C C C C C C4/24 C C C C C C C C C C C4/16 C C C C C C C C C C C4/19 C C C C C C C C C C C5/7 C C C C C C C C C C C5/5 C C C C C C C C C C C5/1 C C C C C C C C C C C5/6 C C C C C C C C C C C5/11 C C C C C C C C C C C5/8 C C C C C C C C C C C5/10 C C C C C C C C C C C5/9 C C C C C C C C C C C5/2 C C C C C C C C C C C5/3 C C C C C C C C C C C5/4 C C C C C C C C C C C5/12 C C C C C C C C C C B1/2 C C C C C C C C C C B1/4 C C C C C C C C C C B1/15 C C C C C C C C C C B1/8 C C C C C C C C C C B1/14 C C C C C C C C C C B1/9 C C C C C C C C C C B1/13 C C C C C C C C C C B1/11 C C C C C C C C C C B1/12 C C C C C C C C C C B1/16 C C C C C C C C C C B1/6 C C C C C C C C C C B1/1 C C C C C C C C C C B1/3 C C C C C C C C C C B1/5 C C C C C C C C C C B1/7 C C C C C C C C C C B2/10 C C C C C C C C C C B2/5 C C C C C C C C C C B2/11 C C C C C C C C C C B2/7 C C C C C C C C C C B2/1 C C C C C C C C C C B2/2 C C C C C C C C C C B2/12 C C C C C C C C C C B2/3 C C C C C C C C C C B2/13 C C C C C C C C C C B2/4 C C C C C C C C C C B2/6 C C C C C C C C C C B2/9 C C C C C C C C C C B3/9 C C C C C C C C C C B3/8 C C C C C C C C C C B3/4 C C C C C C C C C C B3/10 T T T T T T T T T T B3/5 C C C C C C C C C C B3/6 C C C C C C C C C C B3/3 C C C C C C C C C C B3/1 C C C C C C C C C C B3/11 C C C C C C C C C C B3/2 C C C C C C C C C C B3/12 C C C C C C C C C C B4/10 T T T T T C C C C T B4/7 T T T T T C C C C T B4/2 T T T T T C C C C T B4/3 T T T T T C C C C T B4/17 T T T T T C C C C T B4/14 C C C C C C C C C C B4/18 C C C C C C C C C C B4/11 C C C C C C C C C C B4/13 C C C C C C C C C C B4/12 C C C C C C C C C C B4/1 C C C C C C C C C C B4/16 C C C C C C C C C C B4/4 C C C C C C C C C C B4/6 C C C C C C C C C C B4/5 C C C C C C C C C C B4/20 C C C C C C C C C C B4/21 C C C C C C C C C C B4/9 C C C C C C C C C C H1/1 C C C C C C C C C C H1/9 C C C C C C C C C C H1/5 C C C C C C C C C C H1/10 C C C C C C C C C C H1/2 C C C C C C C C C C H1/11 C T C C C C C C C C H1/8 C C C C C C C C C T H1/7 C C T C C C C C C C H1/12 C C C C C C C C C C H1/6 C C C C C C C C C C H1/4 C C C C C C C C C C H2/1 C C C C C C C C C DEL H2/12 C C C C C C C C C C H2/2 C C C C C C C C C C H2/5 C C C C C C C C C C H2/3 C C T C C C C C C C H2/8 C C C C C C C C C T H2/4 C C C C C C C C C C H2/10 C C C C C C C C C C H2/11 C C C C C C C C C C H2/6 C C C C C C C C C DEL H2/7 C C C C C C C C C DEL H2/9 T C C C C C C C C DEL -2951 -2947 -2939 -2935 -2905 -2874 -2860 -2853 -2849 -2844 Clone CG-29 CG-28 CG-27 CG-26 CG-25 CG-24 CG-23 GC-22 GC-21 GC-20 C1/24 C C C C C C C C C C C1/7 C C C C C C C T C C C1/1 C C C C C C C C C C C1/3 C C C C C C C C C C C1/8 C C C C C C C C C C C1/5 C C C C C C C C C C C1/11 C C C C C C C C C C C1/10 C C C C C C C C C C C1/2 C C C C C C C C C C C1/12 C C C C C C C C C C C1/15 C C C C C C C C C C C1/21 C C C C C C C C C C C1/18 C C C C C C C C C C C1/22 C C C C C C C C C C C1/4 C C C C C C C C C C C1/9 C C C C C C C C C C C1/16 C C C C C C C C C C C1/13 C C C C C C C C C C C1/23 C C C C C C C C C C C1/14 C C C C C C C C C C C1/20 C C C C C C C C C C C1/17 C C C C C C C C C C C1/6 C C C C C C C C C C C2/8 C C C C C C C C C C C2/4 C C C C C C C C C C C2/11 C C C C C C C C C C C2/19 C C C C C C C C C C C2/12 C C C C C C C C C C C2/2 C C C C C C C C C C C2/6 C C C C C C C C C C C2/10 C C C C C C C C C C C2/3 C C C C C C C C C C C2/13 C C C C C C C C C C C2/14 C C C C C C C C C C C2/16 C C C C C C C C C C C2/15 C C C C C C C C C C C2/17 C C C C C C C C C C C2/9 C C C C C C C C C C C2/24 C C C C C C C C C C C2/5 C C C C C C C C C T C2/7 C C C C C C C C C C C2/18 C C C C C C C C C C C2/22 C C C C C C C C C C C2/23 C C C C C C C C C C C3/22 C C C C C C C C C C C3/19 C C C C C C C C C C C3/11 C C C C C C C C C C C3/2 C C C C C C C C C C C3/7 C C C C C C C C C C C3/10 C C C C C C C C C C C3/12 C C C C C C C C C C C3/9 C C C C C C C C C C C3/1 C C C C C C C C C C C3/13 C C C C C C C C C C C3/18 C C C C C C C C C C C3/16 C C C C C C C C C C C3/14 C C C C C C C C C C C3/23 C C C C C C C C C C C3/15 C C C C C C C C C C C3/20 C C C C C C C C C C C3/17 C C C C C C C C C C C3/24 C C C C C C C C C C C3/3 C C C C C C C C C C C3/8 C C C C C C C C C C C3/6 C C C C C C C C C C C4/11 C C C C C C C C C C C4/10 C C C C C C C C C C C4/4 C C C C C C C C C C C4/3 C C C C C C C C C C C4/1 C C C C C C C C C C C4/17 C C C C C C C C C C C4/14 C C C C C C C C C C C4/20 C C C C C C C C C C C4/18 C C C C C C C C C C C4/2 C C C C C C C C C T C4/12 C C C C C C C C C T C4/5 C C C C C C C C C T C4/8 C C C C C C C C C T C4/9 C C C C C C C C C T C4/21 C C C C C C C C C T C4/22 C C C C C C C C C T C4/13 C C C C C C C T T C C4/23 C C C C C C C T T C C4/15 C C C C C C C C C C C4/24 C C C C C C C C C C C4/16 C C C C C C C C C C C4/19 C C C C C C C C C C C5/7 C C C C C C C C C C C5/5 C C C C C C C C C C C5/1 C C C C C C C C C C C5/6 C C C C C C C C C C C5/11 C C C C C C C C C C C5/8 C C C C C C C C C C C5/10 C C C C C C C C C C C5/9 C C C C C C C C C C C5/2 C C C C C C C C C C C5/3 C C C C C C C C C C C5/4 C C C C C C C C C C C5/12 C C C C C C C C C C B1/2 C C C C C C C C C C B1/4 C C C C C C C C C C B1/15 C C C C C C C C C C B1/8 C C C C C C C C C C B1/14 C C C C C C C C C C B1/9 C C C C C C C C C C B1/13 C C C C C C C C C C B1/11 C C C C C C C C C C B1/12 C C C C C C C C C C B1/16 C C C C C C C C C C B1/6 C C C C C C C C C C B1/1 C C C C C C C C C C B1/3 C C C C C C C C C C B1/5 C C C C C C C C C C B1/7 C C C C C C C C C C B2/10 C C C C C C C C C C B2/5 C C C C C C C C C C B2/11 C C C C C C C C C C B2/7 C C C C C C C C C C B2/1 C C C C C C C C C C B2/2 C C C C C C C C C C B2/12 C C C C C C C C C C B2/3 C C C C C C C C C C B2/13 C C C C C C C C C C B2/4 C C C C C C C C C C B2/6 C C C C C C C C C C B2/9 C C C C C C C C C C B3/9 C C C C C C C C C C B3/8 C C C C C C C C C C
B3/4 C C C C C C C C C C B3/10 T T T T T T T T T T B3/5 C C C C C C C C C C B3/6 C C C C C C C C C C B3/3 C C C C C C C C C C B3/1 C C C C C C C C C C B3/11 C C C C C C C C C C B3/2 C C C C C C C C C C B3/12 C C C C C C C C C C B4/10 T C T C T C T T T C B4/7 T C T C T C T T T C B4/2 T C T C T C T T T C B4/3 T C T C T C T T T C B4/17 T C C C T C T T T C B4/14 T C T C C C C T C C B4/18 T C T C C C C T C C B4/11 C C C C C C C C C C B4/13 C C C C C C C C C C B4/12 C C C C C C C C C C B4/1 C C C C C C C C C C B4/16 C C C C C C C C C C B4/4 C C C C C C C C C C B4/6 C C C C C C C C C C B4/5 C C C C C C C C C C B4/20 C C C C C C C C C C B4/21 C C C C C C C C C C B4/9 C C C C C C C C C C H1/1 C C C C C C C C C C H1/9 C C C C C C C C C T H1/5 C C C C C C C C C C H1/10 C C C C C C C C C C H1/2 C C C C C C C C C C H1/11 C C T C C C C C C C H1/8 C C C C C T T T T T H1/7 T C C C C T C C C C H1/12 C C C C C C C C C C H1/6 C C C C C T C C C C H1/4 C C C C C C C C C C H2/1 C C C C C C C C C C H2/12 C C C C C C C C C C H2/2 C C C C C C T C C C H2/5 C C C C C C C C C C H2/3 C C T C C C C C C C H2/8 C C C C C C C C C C H2/4 C C C C C C C C C C H2/10 C C C C C T C C C C H2/11 C C C C C C C C C C H2/6 C C C C C C C C C C H2/7 C C C C C C C C C T H2/9 C C C C C C C C C C
TABLE-US-00003 TABLE 3 Non-CpG methylation in CpG-island 4-5 of the MSMB gene ##STR00001## ##STR00002## ##STR00003## ##STR00004## ##STR00005## ##STR00006## ##STR00007## ##STR00008## ##STR00009## ##STR00010## ##STR00011## ##STR00012## ##STR00013##
TABLE-US-00004 TABLE 4 Sequence alterations in CpG-island 4-5 of the MSMB gene ##STR00014## ##STR00015## ##STR00016## ##STR00017## ##STR00018## ##STR00019## ##STR00020## ##STR00021##
TABLE-US-00005 TABLE 5 Summary of all detected haplotypes of CpG island 4-5 of the MSMB gene ##STR00022## ##STR00023##
Sequence CWU
1
771746PRTHomo sapiens 1Met Gly Gln Thr Gly Lys Lys Ser Glu Lys Gly Pro Val
Cys Trp Arg1 5 10 15Lys
Arg Val Lys Ser Glu Tyr Met Arg Leu Arg Gln Leu Lys Arg Phe 20
25 30Arg Arg Ala Asp Glu Val Lys Ser
Met Phe Ser Ser Asn Arg Gln Lys 35 40
45Ile Leu Glu Arg Thr Glu Ile Leu Asn Gln Glu Trp Lys Gln Arg Arg
50 55 60Ile Gln Pro Val His Ile Leu Thr
Ser Val Ser Ser Leu Arg Gly Thr65 70 75
80Arg Glu Cys Ser Val Thr Ser Asp Leu Asp Phe Pro Thr
Gln Val Ile 85 90 95Pro
Leu Lys Thr Leu Asn Ala Val Ala Ser Val Pro Ile Met Tyr Ser
100 105 110Trp Ser Pro Leu Gln Gln Asn
Phe Met Val Glu Asp Glu Thr Val Leu 115 120
125His Asn Ile Pro Tyr Met Gly Asp Glu Val Leu Asp Gln Asp Gly
Thr 130 135 140Phe Ile Glu Glu Leu Ile
Lys Asn Tyr Asp Gly Lys Val His Gly Asp145 150
155 160Arg Glu Cys Gly Phe Ile Asn Asp Glu Ile Phe
Val Glu Leu Val Asn 165 170
175Ala Leu Gly Gln Tyr Asn Asp Asp Asp Asp Asp Asp Asp Gly Asp Asp
180 185 190Pro Glu Glu Arg Glu Glu
Lys Gln Lys Asp Leu Glu Asp His Arg Asp 195 200
205Asp Lys Glu Ser Arg Pro Pro Arg Lys Phe Pro Ser Asp Lys
Ile Phe 210 215 220Glu Ala Ile Ser Ser
Met Phe Pro Asp Lys Gly Thr Ala Glu Glu Leu225 230
235 240Lys Glu Lys Tyr Lys Glu Leu Thr Glu Gln
Gln Leu Pro Gly Ala Leu 245 250
255Pro Pro Glu Cys Thr Pro Asn Ile Asp Gly Pro Asn Ala Lys Ser Val
260 265 270Gln Arg Glu Gln Ser
Leu His Ser Phe His Thr Leu Phe Cys Arg Arg 275
280 285Cys Phe Lys Tyr Asp Cys Phe Leu His Pro Phe His
Ala Thr Pro Asn 290 295 300Thr Tyr Lys
Arg Lys Asn Thr Glu Thr Ala Leu Asp Asn Lys Pro Cys305
310 315 320Gly Pro Gln Cys Tyr Gln His
Leu Glu Gly Ala Lys Glu Phe Ala Ala 325
330 335Ala Leu Thr Ala Glu Arg Ile Lys Thr Pro Pro Lys
Arg Pro Gly Gly 340 345 350Arg
Arg Arg Gly Arg Leu Pro Asn Asn Ser Ser Arg Pro Ser Thr Pro 355
360 365Thr Ile Asn Val Leu Glu Ser Lys Asp
Thr Asp Ser Asp Arg Glu Ala 370 375
380Gly Thr Glu Thr Gly Gly Glu Asn Asn Asp Lys Glu Glu Glu Glu Lys385
390 395 400Lys Asp Glu Thr
Ser Ser Ser Ser Glu Ala Asn Ser Arg Cys Gln Thr 405
410 415Pro Ile Lys Met Lys Pro Asn Ile Glu Pro
Pro Glu Asn Val Glu Trp 420 425
430Ser Gly Ala Glu Ala Ser Met Phe Arg Val Leu Ile Gly Thr Tyr Tyr
435 440 445Asp Asn Phe Cys Ala Ile Ala
Arg Leu Ile Gly Thr Lys Thr Cys Arg 450 455
460Gln Val Tyr Glu Phe Arg Val Lys Glu Ser Ser Ile Ile Ala Pro
Ala465 470 475 480Pro Ala
Glu Asp Val Asp Thr Pro Pro Arg Lys Lys Lys Arg Lys His
485 490 495Arg Leu Trp Ala Ala His Cys
Arg Lys Ile Gln Leu Lys Lys Asp Gly 500 505
510Ser Ser Asn His Val Tyr Asn Tyr Gln Pro Cys Asp His Pro
Arg Gln 515 520 525Pro Cys Asp Ser
Ser Cys Pro Cys Val Ile Ala Gln Asn Phe Cys Glu 530
535 540Lys Phe Cys Gln Cys Ser Ser Glu Cys Gln Asn Arg
Phe Pro Gly Cys545 550 555
560Arg Cys Lys Ala Gln Cys Asn Thr Lys Gln Cys Pro Cys Tyr Leu Ala
565 570 575Val Arg Glu Cys Asp
Pro Asp Leu Cys Leu Thr Cys Gly Ala Ala Asp 580
585 590His Trp Asp Ser Lys Asn Val Ser Cys Lys Asn Cys
Ser Ile Gln Arg 595 600 605Gly Ser
Lys Lys His Leu Leu Leu Ala Pro Ser Asp Val Ala Gly Trp 610
615 620Gly Ile Phe Ile Lys Asp Pro Val Gln Lys Asn
Glu Phe Ile Ser Glu625 630 635
640Tyr Cys Gly Glu Ile Ile Ser Gln Asp Glu Ala Asp Arg Arg Gly Lys
645 650 655Val Tyr Asp Lys
Tyr Met Cys Ser Phe Leu Phe Asn Leu Asn Asn Asp 660
665 670Phe Val Val Asp Ala Thr Arg Lys Gly Asn Lys
Ile Arg Phe Ala Asn 675 680 685His
Ser Val Asn Pro Asn Cys Tyr Ala Lys Val Met Met Val Asn Gly 690
695 700Asp His Arg Ile Gly Ile Phe Ala Lys Arg
Ala Ile Gln Thr Gly Glu705 710 715
720Glu Leu Phe Phe Asp Tyr Arg Tyr Ser Gln Ala Asp Ala Leu Lys
Tyr 725 730 735Val Gly Ile
Glu Arg Glu Met Glu Ile Pro 740
74522695DNAHomo sapiens 2caaataaaag cgatggcgat tgggctgccg cgtttggcgc
tcggtccggt cgcgtccgac 60acccggtggg actcagaagg cagtggagcc ccggcggcgg
cggcggcggc gcgcgggggc 120gacgcgcggg aacaacgcga gtcggcgcgc gggacgaaga
ataatcatgg gccagactgg 180gaagaaatct gagaagggac cagtttgttg gcggaagcgt
gtaaaatcag agtacatgcg 240actgagacag ctcaagaggt tcagacgagc tgatgaagta
aagagtatgt ttagttccaa 300tcgtcagaaa attttggaaa gaacggaaat cttaaaccaa
gaatggaaac agcgaaggat 360acagcctgtg cacatcctga cttctgtgag ctcattgcgc
gggactaggg agtgttcggt 420gaccagtgac ttggattttc caacacaagt catcccatta
aagactctga atgcagttgc 480ttcagtaccc ataatgtatt cttggtctcc cctacagcag
aattttatgg tggaagatga 540aactgtttta cataacattc cttatatggg agatgaagtt
ttagatcagg atggtacttt 600cattgaagaa ctaataaaaa attatgatgg gaaagtacac
ggggatagag aatgtgggtt 660tataaatgat gaaatttttg tggagttggt gaatgccctt
ggtcaatata atgatgatga 720cgatgatgat gatggagacg atcctgaaga aagagaagaa
aagcagaaag atctggagga 780tcaccgagat gataaagaaa gccgcccacc tcggaaattt
ccttctgata aaatttttga 840agccatttcc tcaatgtttc cagataaggg cacagcagaa
gaactaaagg aaaaatataa 900agaactcacc gaacagcagc tcccaggcgc acttcctcct
gaatgtaccc ccaacataga 960tggaccaaat gctaaatctg ttcagagaga gcaaagctta
cactcctttc atacgctttt 1020ctgtaggcga tgttttaaat atgactgctt cctacatcgt
aagtgcaatt attcttttca 1080tgcaacaccc aacacttata agcggaagaa cacagaaaca
gctctagaca acaaaccttg 1140tggaccacag tgttaccagc atttggaggg agcaaaggag
tttgctgctg ctctcaccgc 1200tgagcggata aagaccccac caaaacgtcc aggaggccgc
agaagaggac ggcttcccaa 1260taacagtagc aggcccagca cccccaccat taatgtgctg
gaatcaaagg atacagacag 1320tgatagggaa gcagggactg aaacgggggg agagaacaat
gataaagaag aagaagagaa 1380gaaagatgaa acttcgagct cctctgaagc aaattctcgg
tgtcaaacac caataaagat 1440gaagccaaat attgaacctc ctgagaatgt ggagtggagt
ggtgctgaag cctcaatgtt 1500tagagtcctc attggcactt actatgacaa tttctgtgcc
attgctaggt taattgggac 1560caaaacatgt agacaggtgt atgagtttag agtcaaagaa
tctagcatca tagctccagc 1620tcccgctgag gatgtggata ctcctccaag gaaaaagaag
aggaaacacc ggttgtgggc 1680tgcacactgc agaaagatac agctgaaaaa ggacggctcc
tctaaccatg tttacaacta 1740tcaaccctgt gatcatccac ggcagccttg tgacagttcg
tgcccttgtg tgatagcaca 1800aaatttttgt gaaaagtttt gtcaatgtag ttcagagtgt
caaaaccgct ttccgggatg 1860ccgctgcaaa gcacagtgca acaccaagca gtgcccgtgc
tacctggctg tccgagagtg 1920tgaccctgac ctctgtctta cttgtggagc cgctgaccat
tgggacagta aaaatgtgtc 1980ctgcaagaac tgcagtattc agcggggctc caaaaagcat
ctattgctgg caccatctga 2040cgtggcaggc tgggggattt ttatcaaaga tcctgtgcag
aaaaatgaat tcatctcaga 2100atactgtgga gagattattt ctcaagatga agctgacaga
agagggaaag tgtatgataa 2160atacatgtgc agctttctgt tcaacttgaa caatgatttt
gtggtggatg caacccgcaa 2220gggtaacaaa attcgttttg caaatcattc ggtaaatcca
aactgctatg caaaagttat 2280gatggttaac ggtgatcaca ggataggtat ttttgccaag
agagccatcc agactggcga 2340agagctgttt tttgattaca gatacagcca ggctgatgcc
ctgaagtatg tcggcatcga 2400aagagaaatg gaaatccctt gacatctgct acctcctccc
ccctcctctg aaacagctgc 2460cttagcttca ggaacctcga gtactgtggg caatttagaa
aaagaacatg cagtttgaaa 2520ttctgaattt gcaaagtact gtaagaataa tttatagtaa
tgagtttaaa aatcaacttt 2580ttattgcctt ctcaccagct gcaaagtgtt ttgtaccagt
gaatttttgc aataatgcag 2640tatggtacat ttttcaactt tgaataaaga atacttgaac
ttgtcaaaaa aaaaa 26953572DNAHomo sapiens 3gtacctgtct ataaggagtc
ctgcttatca caatgaatgt tctcctgggc agcgttgtga 60tctttgccac cttcgtgact
ttatgcaatg catcatgcta tttcatacct aatgagggag 120ttccaggaga ttcaaccagg
aaatgcatgg atctcaaagg aaacaaacac ccaataaact 180cggagtggca gactgacaac
tgtgagacat gcacttgcta cgaaacagaa atttcatgtt 240gcacccttgt ttctacacct
gtgggttatg acaaagacaa ctgccaaaga atcttcaaga 300aggaggactg caagtatatc
gtggtggaga agaaggaccc aaaaaagacc tgttctgtca 360gtgaatggat aatctaatgt
gcttctagta ggcacagggc tcccaggcca ggcctcattc 420tcctctggcc tctaatagtc
aatgattgtg tagccatgcc tatcagtaaa aagatttttg 480agcaaacact tgaatatgtg
tgtcctttta atttataatt tatgtatgca ttgatttaat 540acacattata aagaatacaa
aacatttaaa aa 5724420DNAHomo sapiens
4gtacctgtct ataaggagtc ctgcttatca caatgaatgt tctcctgggc agcgttgtga
60tctttgccac cttcgtgact ttatgcaatg catcatgcta tttcatacct aatgagggag
120ttccaggaga ttcaaccagg atgtttctac acctgtgggt tatgacaaag acaactgcca
180aagaatcttc aagaaggagg actgcaagta tatcgtggtg gagaagaagg acccaaaaaa
240gacctgttct gtcagtgaat ggataatcta atgtgcttct agtaggcaca gggctcccag
300gccaggcctc attctcctct ggcctctaat agtcaatgat tgtgtagcca tgcctatcag
360taaaaagatt tttgagcaaa cacttgaata tgtgtgtcct tttaatttat aatttatgta
420530000DNAHomo sapiens 5ctctaaaact aaaaacaatg tttgtcagga gttacaaacc
atgaccaact aattatgggg 60aatcataaaa tatgactgta tgagatcttg atggtttaca
aagtgtaccc actgttaatc 120actttaaaca ttaatgaact taaaaatgaa tttacggaga
ttggaatgtt tctttcctgt 180tgtattagtt ggctcaggct gccataacaa aataccacag
actgggaggc ttaagtaaca 240gaaattcatt tctcacagtt ctgggggctg gaagtccacg
atcaaggtgc aggaaaggca 300ggcttcattc tgaggcccct ctcttggctc acatgtggcc
accctcccac tgcgtgctca 360catgacctct ttgtgctcct ggaaagaggg tgtgggggac
agagggaaag agaaggagag 420ggaactctct ggtgtctcgt ctttcaagga ccctaacctg
ggccactttg gcccaggcac 480tgtggggtgg ggggttgtgg ctgctctgct ctgagtggcc
aagataaagc aacagaaaaa 540tgtccaaagc tgtgcagcaa agacaagcca ccgaacaggg
atctgctcat cagtgtgggg 600acctccaagt cggccaccct ggaggcaagc ccccacagag
cccatgcaag gtggcagcag 660cagaagaagg gaattgtccc tgtccttggc acattcctca
ccgacctggt gatgctggac 720actgcgatga atggtaatgt ggatgagaat atgatggact
cccagaaaag gagacccagc 780tgctcaggtg gctgcaaatc attacagcct tcatcctggg
gaggaactgg gggcctggtt 840ctgggtcaga gagcagccca gtgagggtga gagctacagc
ctgtcctgcc agctggatcc 900ccagtcccgg tcaaccagta atcaaggctg agcagatcag
gcttcccgga gctggtcttg 960ggaagccagc cctggggtga gttggctcct gctgtggtac
tgagacaata ttgtcataaa 1020ttcaatgcgc ccttgtatcc ctttttcttt tttatctgtc
tacatctata atcactatgc 1080atactagtct ttgttagtgt ttctattcaa cttaatagag
atatgttata cttaaaaaaa 1140aaaaaaaaat gggccgggcg cagtggctca tgcctgtaat
cccagcactt tgggaggccg 1200aggtgggtgg atcatgaggt caggagatcg agaccatcct
gattaacgtg gtgaaacccc 1260gtctctacta aaaatacaaa aaaaattagc cgggcgtggt
ggcaggcgcc tgtagtccca 1320gctactcagg aggctgaggc aggagaatgg cgtgaacccg
ggaggcagag cttgcagtga 1380gccgagatgg cgccactgca ctccagcctg ggtccagcct
gggcaacaag agtaaaaccc 1440tgtctcaaaa aaaaaaaaaa aaagataaat aataaaaaat
aaaatataaa taaaaaagca 1500aactgcagac tctcagcaca aatgatttca tccacctctg
gcactaagag acttcaacaa 1560acactgttgt ttctaacagt tcatggccac atccttagga
tgatgacttc agccccctta 1620agtttctgcc taataaaatt caatgctgcc caaagaattt
atgatttgtt ccaggcaaaa 1680cctgaatata ggcccccgaa cccccttttc ctaaaggatt
tactttagaa aactggcagt 1740tagaaatcct ttctctgtct cttggaaatg tatcttctat
aacacaggaa tgtctttccc 1800aaggacctgg gagccatcct ttcgaaatgt aatcattagg
taggacagag cctctgtctc 1860ccaatcttgg tggtagggta ggaccctgat attgataagc
accagttcac aatttgataa 1920gcacaagttc acaatctggt cagtgtactc attgaccaaa
cctccctccc ctcaactagc 1980atccttcagt acttttccct taacacatcc tagcatttaa
aaagcctctt gccttctggt 2040ttggtagaat tgagctcagt taatgctgaa gtgtctcttc
cctgccacag tagtctgaat 2100aaaatctgtc ttgacatttt tgacaagtgt ctggtgcaaa
atttttcttt aacaatagca 2160agtgttgaaa ttggatagtg taagtttttt ttttagcttt
gttctttttc agaatattta 2220ggctatccta ggtcattcgc ttttctatat aaattttagc
aaaagcttat caatttccac 2280aaaaacctgc tggactattt tttttgtttt tgtttttgag
accaagtctc actctgtcat 2340ccaggctgga gtgtggtggc acaatcttgg ctcactgcaa
cctccgcctc ccaagttcaa 2400gcgattctcc tgcctcagcc tccctagtag ctgggattac
aggcagatgc caccatgccc 2460agataatttt tgtattttta gtagagtcac tgtttcacca
tgttggccag gctggtcttg 2520aacacctgaa ctcaggtgat ccacctgcct cggcctccca
agtgctggga ttacaggcgt 2580gagccactgc acccagccag gactgacttt ttaatttttt
cccttgattt cgcagtattt 2640ggaatggact cactttctta ggaacgacat taattttttt
taaattggca gaaatttatc 2700aataaattgg gggaggactg tcatcttaac aatagagtct
tttaatctat gaacatggga 2760ttatttaaat cagggattag caaattttct gcaaagaacc
agatagtaaa aatatttgtg 2820gtattgtggg ctatattcaa ctttgctttg tagtgtaaaa
gcagtgttgg cccggcgcgg 2880tggctcatgc ttgtaatccc agcactttgg gaggctgagg
cgggtggact acctaaggtc 2940aggagttcaa gaccagctgg ctaacatgga gaaaccccgt
tactactaaa aatacaaaaa 3000aaatagccag gcatagtggc gcacgcctgt aattccaact
attcgggagg ctgagacagg 3060agaatcgctt gaacctggga ggtggatgtt gcagtgagct
gagatcacac cattgcactc 3120cagcttgggc aacaagagca aaactccatc tcaaaaaaaa
aaaaattgtg tttcaataaa 3180actttattta caaaacaaac agggggccag ttcagggcca
tggactgtag tttgctgcct 3240ctaatttaga acttcttcaa tttctctgag caaagtttta
tagtttttat tgtaagtctt 3300gcatttcttt cattaaactc atttctaagt attttattct
tttaggtgtt actgtgaatg 3360aaattttgtt tccttaattt cattttcaca ttgtttatta
ctagtatgta gaaatacaat 3420tgatttttac atatcggcct tatatacttg aaccttgttt
gctaaactca tttattaatt 3480ccagtaaaat tttttgatag attacttggg attttctgca
tacagatcat gtggtctaca 3540aataaacaca gttttacttt tttcttttca ttctagatgt
ctttaattta cctttctctc 3600tctttctccc tctaccccct ttttaagttg ccctggctat
gaccagtagt acaatgttga 3660atggaagtgg taagagtaga cattcttgcc tgtttcctaa
tcttgagtgg gaaacattct 3720gtctttcatc attaagtaca ttaactgtag gttgtctgta
gttgccctct atcagattga 3780agaagtttac ttttattcct agtttgttga gagtttttac
cacgaatgta tgttggctat 3840tgtcaaatgc ttctgtatct attgaaatga caatgtgatg
tattttagtc tattaatcta 3900gtacattaca ttcactgatt ttgaggtttt gttgttttta
attaattaat tttaaaaacc 3960tcaccatata ctagagaaag cattacttga tttttggatg
tcacaccaac cttgcatttc 4020tgggataaat catacttggt catggtgtat aatcttttgt
acatgttgat ggatttttgt 4080ttgctaatat cttattgagt atgtttgcat ctctattcat
gagagatatt gttctataat 4140ttttgtttat tttggtgtgt ttgtttagct ttgatattgg
ggtaacactg gcctcattaa 4200aggagttgga acatgatcta tcctttttta ttttctgaaa
gatttttgtg aaagacataa 4260tatttcttct cgtcttattg tgtcgtgtcg tgtcctgtcc
tgtctttttg aggtggagtt 4320ttgctcttgt tgcccaggcg ggagtgcaat ggcatgatct
cggcccattg caacctccac 4380ctcccaggtt caggtgatgc tcctgcctcc caagtagctg
ggattacagg tatgcaccac 4440cagacctggc taattttttt tttttttttt tttttgagac
aaagtcttgc tctgtcaccc 4500aggcgggagt gcagtaacgt gatcttggct cactgcaagc
tccgcctccc gggttcacgc 4560cattctccta cttcagcctc ctgaatagct gggactacag
gcacccgcga ccacacctaa 4620ctttttttgt atttttagta gagacggggt ttcaccacgt
tagccaggat gatctcgatc 4680tcctgacctc atgatccacc cgtctcggcc tcccaacgtg
ctgggattac agttatgagc 4740caccatgccc tgcctaattt tgtattttta gtagtgatgg
ggtttcacca tgttgatcag 4800gctggttttg aactcctgac ctcaggccat ccacccgcct
ttgcctccaa tttggctaat 4860ttttttattt tgtagagatg gagttctcac tgtgttgtcc
aggctgctct tgaatttctg 4920gcctcaagca atcctcctac ctgggcctcc caaagttctg
ggattatagg tataagccac 4980tactcctggc ctaaaaatgt attttttata catatgtctc
tgtcaaaatt gggataaaga 5040gaaacagact ggcagtggta tgcatttcaa ggaaaatagg
agaaagccta tttacttagg 5100aaagtgaggc tcataagtac ttcatataca aacatcatac
ctgcttcact gggttgtgtt 5160acctgacttg ccccgagcat ttgaacttgc tacccccaat
agagaattga ctccctcatt 5220agccaaagag gaatctgaga cctggtgagg gtgagcacct
ggccagtcat cagtaagctc 5280cttttcatcc cacccaccgg cggtgattag taaccatgtc
tgacaggcat ttactttcct 5340cctggtgagt agctggaaaa gagagtagtt tggaaatatt
tggaaatagc accgtttgtc 5400tctcacatat gtgatctgga agccccctgc ctgcttcaca
ttgtcctgcc tttccagact 5460gaaccagtgt tcattttaca tatgttgagt gatggctcat
gtctccctaa aaggtataaa 5520accaagctgt gctctgacta ccttgggcac atgttgtcag
aacctcctga gggtgtgtca 5580cggtagcata tcctcaacct tggcgacttg gtgctaacgc
tgtactaaat taccaggctg 5640gttaacatct attggaattt cttggtgcca aaggctgtgg
gagacctgaa cacattgtca 5700agggaggagg aaggggaagg agaagggctg tgggatctcc
cgcatgaaag gccccatgct 5760ggtggtagcc cccacctgct gctctggggc tcaaaggtgc
ttagatctct tcacttgagt 5820gcagatgctg ccagcccttt gggcaggtgt gtgagggagg
cagccctggc tgtaaagaga 5880gcaggggctt ggctgggcgt ggtggctcac gcctgtaatc
ccagcacttt gggaggccga 5940ggtggcagat catttgaggt caggagtttg tgaccagcct
ggccaacatg gtgaaacccc 6000atctctacta aaaaaaaaat acagaaatta gctgggcatg
gtggcatgta cctgtaatcc 6060cagctacgca ggaggctgag gcaggagaat cacttgaacc
caggaggcgg aggttgcagt 6120gagctgagat agcgccactg cactccagcc cgggcgacag
cgcgagactc catctcaaaa 6180aaaaaaaaaa aaaaaaaaaa aagagagtac tggcttttgg
gtcattagga ctattatata 6240actgaattgt gtcaggcctc tgagctgaag ctcagctatt
ataacccctg tgacctgcac 6300atatatgtcc agatggcctg caggaaccaa gaagtctgga
gcagccaaaa aacccacaaa 6360gtaaaacagc cagttcctgc cttaactgag taaccaaaat
tacaacattt taccattgtg 6420acttgtccct gccctacctt agctgatcaa ccaactttgt
gacattcttc ttctggacaa 6480tgagtcttat gatctcccca ctatgtacct tgtgaccccc
tcctctgcta acaatagata 6540accacgtttt actgtaactt tccattacct acccaactcc
tataaagcaa tcccttcccc 6600atctcccttt gctgactctt ttcggactca gcccacctgc
acccaagtga attaaaagtt 6660ttattcctca tacaaagcct gtttggtggt ttcttcacat
ggacgtgctt gacaaattgg 6720ggtcggcttg cccagtgcag ttaagtccag atatccacac
tgaagttgca gcactagaaa 6780ggaaggtgtt tattcacaga gccctgagca agggggacca
ggcagcgcat gctcaaaacc 6840tgaactctct gatggctggc agatgtgaac cctgaaaatc
tgagacaagt gtcaatttag 6900aaagtttatt ttgccaaggt tgaggacatg cccccgtgac
acaccctcag gaggttctga 6960cgacatgtgc ccaaggtggt cagagcacag cttggtttta
taccttttag ggagacatga 7020gccatcaatc aacatatgta aaatgaacat tggtttggtc
tggaaaggca ggacaacttg 7080aagcaggcag tgggcttcca gatcacagat aggtgagaga
caaacggttg cattcttttg 7140agtttctgat tagcctttcc aaaggaggca atcagatatg
catttatctc agtgagcaga 7200gggatgactt tgaatagaat gcgaggcaga tttgccctaa
gcagttccca gcttgaattt 7260tctctttaac ttagtgattt gggagggcca acatagtttc
ctttcacaca ggtaagggtt 7320tttaaaggcg gggtaaattt tcaggaaagc agcagaagtt
agataaaatt gtaaatcagt 7380acatgaaggg tacacattgg tttggcctaa aaaaggtgag
atatcttgaa gcaggggctt 7440acaggtcata ggtggattca gagattcttt gatttgtagt
tggttcagga ggcgatgcct 7500tgcctgaaaa ttcagggtta acagaaaaga atattagctt
tagctcatgg gtgtggcttt 7560caggagcctc cggaagaaat ttagaatgaa agaaggtggt
cagagtgcag tcctcagctc 7620cctctatctg aggtctgcag gccagcagat ccgtttggtg
ggggtccaca tggaggtcca 7680ggtttctgaa tgacaaactc aaggacgtgt gttaagatgt
tatctttagt tcctaaaact 7740aaacatctcc ggactctaac ttccttggct attgttttgg
gctactatta ccttcctgcc 7800taacaagtta cttacttact tctcagggct agctggatgc
ctgaaatttc ccttgaaggc 7860actcaagatt ttcctttatt tccatgcttg atggtggcgt
tcggggtggt ggcacaggtc 7920cctgcttcat ctcaggcctg ggtttaaatt ctcactcttt
ctcaccatct gaaaaccctg 7980aatagttgtt tccatcctga aacctcaatg ccccactggt
aaagtgggcg agagaaggcc 8040tgttttgttg tgttgctggg aaatccctga ggtaatgcat
gtgtggacat gagaggctcc 8100agctcccttc ccacctctca gccctccacc ctggggtggt
tctggaaggc actgctcatg 8160gtaaggacag aaggttgctg gctggcgttc ttctgtctga
ttcagtggct gcatttactt 8220agcatgtgct ctatgaggag aataagggcc tgtgatgctg
gtatagatca gttcagatat 8280actggtgggt acgtggctgc tgcccaacac tcctgagtgc
ccccactcag catgttggat 8340cctggcccct ccagacctca acatcatcca gaaacagaag
gaatagagcc tcgcacagta 8400gggtccccca gactgacttc tggcagctga actgaagggt
cagtggttga gccatactgg 8460ttgttcaaca ctttgaatac cacatccaga aagaaaatac
aagctccctg tcacggtagc 8520ctccttgtga ctgtgttaca ttcaacacaa gacaaacatc
agcttcacaa aagggagcag 8580tctctgggac aatccattga aagcgtgaga ggcaggtagt
tcctgggttc tgtgcatagt 8640tgctttaagc tgtaaacaca caatccttga ccaaataccc
tttaatccac ttgacaaaca 8700agaagcttcc gggcctccaa ggggatgccc aagtctccct
tttctctggg acaaaacctg 8760gttttgtttc ttaggtttct cagcggctaa tctggaacat
gtttgctttt gccccgtttc 8820tttgtcccag tgttttttct gctgttttca cacctctctg
gataagcttt ccaagttcct 8880tacagaaaac cagaaagatt attcaccagc agtcttcctg
gtccctggct tgtcagcctc 8940aatgcattcc agtgcactct tgtgacactg tatccagagt
agctgtggct cacagcattt 9000acagtgttct ttctcgctat tctattagtg gcttcctttt
ccaacccagt tctttgcctg 9060gatatccagg atctctatcc caccgtcacc ctcactccat
atagggggtt catcctacag 9120catttctgtt gctgtaaaaa gaccctctac tgcatccttt
ggcatggaga atttgtctta 9180ttgtattttt gaagcaagag attatgcaga aatgaatcca
tgggtggggg cagagtctgt 9240ctttctggtt tctgcagagt gaaaactctc ataatgtctg
tcctagacta agccccgcag 9300tccagaagct ggtccacctc tggacttaac acttgagctg
aaaacgtctc tttttgccca 9360tgctcatttt agttcggttt ccaacagaac agaatcctga
cgaataccaa aacgggtcct 9420gccacagggt atcacaaaga ttgggctcta caaagtaaaa
ttgccagggt ataagtggac 9480cctcagggag gctggcttat gccaggctgg gaattgggca
gtcctcgaac actgaacact 9540tttccatctg tcccttcatg cctgcttgtc ttggggaggc
tggcttttat ggaggggtcc 9600ctgggaagga atgggggccg atggggaggg gaatgggatc
cgtattgcgt atttggggtg 9660aggaagggac ccagagggaa agtggaaaga acaggagccg
gtggagggca atgtgcaggg 9720taggggtggg ggtgcagttt tcacctctgg tctctgtgag
atacgaaata agattgtgtt 9780ctgcgtatga agtaggcagt tgggtgagag gtttaagaga
agagatcatg gtttggaaag 9840gctctagagc acgatgggag aggaagtgga tgagagataa
gtcatcagct tgaagactgg 9900taaggggaga ccccaccgat cctggtgtta aatcaagttt
agcctaaagc cttctcctta 9960catattttaa attcacccta atggtttctc tgtacatagt
gaactgtaag ctaactgcac 10020gtgtaaacag gctgtcacct actctcgtac caagtagccg
agtctcagtt aatcacagca 10080gccagacttc aaccactcac aggcggccag ctgttgaaac
tggactcaaa taagccaagc 10140accatgccat caccaatccg tctgtttctg tacctcactt
ccagtttctg tacatcactt 10200tcctttttct gtccataaat ctttgactat gaggcagtgc
aggagtctct ctgaacttat 10260ttttgcctgg gggctgcctg attcacacaa atggttcttt
gctcaatcaa actctgttaa 10320atttaatttg tctaacgttc ttttaacact gggaatgatg
gcagggcacg gtggcccatg 10380cttgtaatcc cagcactttg ggagaccaag gcaggcagat
cacttgaggt caggcgttca 10440agaccaacct gggcaatggt gaaaccctgt ctctactaaa
aatacaaaaa ttagccaggc 10500atggtggtgc atgcctatag ccccagctac tgggaaggct
gaagcacaag aatcgcttgt 10560gcccgggagg tggaggttgc agtgagctga gattgcagca
ctgcactcca tcctgggcga 10620cagagcaaga ctctgtctca gaaaaaaaca aaaaacaaaa
caaaacaaaa aaccctggaa 10680atgaatggca gctcctgcca cagcatcagc ccaagggctg
tgtgactcca tggatcttga 10740ggtgttcttt ggggcttgta gtgctgaccc acaggtgggg
atagtgtggc tgtagtcagt 10800taaagtttaa aatctctaca tgtcaaaaca tatcatttga
gaggtaaaaa tataaatgag 10860gaaaatttct cattccttag ttctaactcc agaatgggtg
tgctgaccat gaaaaaaatc 10920agggaaatat tttaacatgt gtgacacttg aagattaata
tctttaatat tagaagcctt 10980aactaatcca gaagaaaaat ataaataacc caaacagaaa
aatagctaaa ggacatgagt 11040gaacagggta ctcacaaaag aagaaaaaca attggtcaat
acatatacaa aaaaaggtct 11100aggctgtgca cggtggctca cgcctgtaat cccagcactt
tggaagcccg aggggggcag 11160atcacgaggt caggagatca agaccatcct gactaacatg
gtgaaacccc gtctctatta 11220aaaatacaaa aaattagccg ggtgtgatgg agggtgcctg
tagttccagc tactcgggag 11280gctgaggcag gagaagggcg tgaacctgga aggtggagct
tgtactgagc tgaaattgca 11340ccattgcact gcagcctggg agacagagcg agactccgtc
tcaaaaaaaa aaaaaaaaaa 11400aaaaaaaaag gtctaatctt gccaattagc aaagaaaaat
aaattaagac aaagcatttc 11460ccacatatca tagtaacagg tgataatcta agagaattat
aattttcttt tacccagaaa 11520ttctaccttt gtgatttttt ttttttttga gatggagttt
cgctcttgtt gcccaggttg 11580gagtgcaatg gcgtgatctc agctcacagc aaccttcgtc
tcccggtttc aagtgattct 11640cctgcctcac cctccggagt agctgggatt acaggcatgc
tccaccacgc ccggctaatt 11700ttgtattttt agtagagacg gggtttctcc atgttggtca
ggctggtctc gaactcgtga 11760cctcacgtga tccacccacc tcgtccttcc aaagtgctgg
gattacaggt gcgagccacc 11820tcacccggcc tctatgaatt tatcttaaag aaattagtat
aagtgtggga aatttttatc 11880tgaaggacct ggatttcaac agcgtttctt tttttttttt
ttgagacaga gtctcgctct 11940ctcgcccagg ctggagtgca gtggcgcgat ctccgctcac
tgcaagctcc gccttccggg 12000ttcacgccat tctcctgcgt cagccttccg agtagctggg
actacaggcg cccgccatga 12060cgcccggcta atttttttgt atttttagta gagacggggt
ttcaccatgt tagccaggat 12120ggtctcgatc tcctgacctc gtgatccgcc cgtctcggcc
tcccaaagtg ctgggattac 12180aggtgtgagc caccatgccc agcccagtgt ttctaattat
agaaaaaaaa gtcagaaaca 12240acctgagtgt tcaacaagag gtacctgacc aaataaatga
tggtccatat cattccatgg 12300aataaaatgt agccattaaa atgataatca gcctgggcat
ggtggcttat gcctgtaatc 12360ccaacacttt gggaggagga ggcaggagca tcacttgagc
ccaggagttc aagaccaggc 12420tgggcagcat agcaagaccc tgtctctaca aaaaaaatct
aaaaattagc tgggtgtggt 12480ggcacgctcc tgtagtccca cctacttggg aggcttaggt
gggaggatca attgaatcta 12540ggaattggag gtaacagtga gctataatgg tgccactgca
ctccagcctg agcagcagag 12600caagactatg tctttaaaaa aaattaaaat aaataaaaag
aaaaataaat aattaaaaga 12660aattaagttg gttccatcta taaggatgac agttgaggta
ggatcaacac tgaaaactga 12720tcaggcaggt ttggacagag ctggctgtca cacacccgga
gagcactgct catttcattt 12780gccttcctcc ctgtgggcag ctggccacac ctgctctgag
catccctttc tcaacccctg 12840gggagcagta gatgagctca tttcagcagt gatatgggtt
ggtcttgcca ggatactttc 12900tacttgggaa tacctgagat aacccaggtg aaatcctgtt
ctgggcagct gctttgcacc 12960aggcaccctg ctggtcactg gaaggcacac agacaggaat
aggtcacagt cactgtctgg 13020gtcacaaagt tcttaactgc aaacaataga atttactcta
ggtcatttga ggtggaaaga 13080gatttgttag agaattcaag cctctactaa cggccagaga
cacaggcttt cttggcacaa 13140gttcaggtag agcactgaaa ccacactgct gcttggctcc
agcaagacca ccttgctacc 13200agtcacagtc acagccccca tgcactgaca ctgcacactg
gactccaaga aacccttgcc 13260ctaagagcca gaaccctcca caccatcatt cctagggaga
ttctgcatga tgccagtgtc 13320cttggaccct ctctgaagag gactttttga gaggagagtg
tttgtggagc cgaggtttgt 13380gcctgtaccc tggaggtaag caggtgaggg cagcaaatcc
tagttcctct caatgtaagg 13440cagaacttcc cacttcacac tgtgggaagt tcttcaaact
tggagtttta aaagaatcca 13500aggggctaca gtgcatgatg aatgtgcccc gatttcctgg
aacttagagt cgatgaacac 13560ttaacttatg caggtaaagg cagaagtgtg tggcaccgtt
ggtgagtgag gaggcagcca 13620gacagcacta cagctcttct aaggagcagg agagtgacct
caagctgtgt taggggtggt 13680gaggctgatt ttagggaggg tagaggatgg tgctggaaag
ggccactgtg gccagctcag 13740atgagcttca aggatcccct ggttaggagc atgaactctc
atctgtgggc agagggatct 13800caggttaaat tttaggaact gtagcaagct gtggcgagga
gtgaggggac tttttgggat 13860aaaattgtct tatgagctca attgcatccc ctaaaaattc
attggtttaa gccctaaccc 13920ccaatatccc agaatgtgac tgtaatggag acacggcctt
taaagaggtg gcttaggtaa 13980aatgagatag ttagagtgga ccctaatcca atacagctag
tgtccttatc agaagaggaa 14040atttggacac agagagagat gccagggatg catgccatgt
aagaggcagc aagagcatgg 14100ccatctgcaa accaaagaga gagggctcag aggaaaacaa
gccttcccca atggcttgac 14160ctcacatttc tagtctccag aactgtgagc aaataaattt
ctttagtcaa agccacccag 14220ttgcagtgtt ttgttgtggt ggcagagcaa acagataaag
tagtaagtcc ctccagcacc 14280ctaggaggtt ctgcttgttg ccattcccca aaggccatcc
tagccatgat gagggcaggt 14340gcggttgttt gggaggagac cactgtggaa caaagagaaa
caaggcttgc ttgatgcagc 14400agggagtaga ctacagattc aagaagggct ctagaaaggt
ccagcaattc agcttgtcag 14460ggaaccccga gctgtatgta gttcttgctc tcctgtgccc
tgcaggatgg cttgacccat 14520ttctgttctc atgtcagacc ctaagggcag ggtcatctgg
gccacagacc caggcccggt 14580gggcctggtg ggctgacctg tgggctattc ttatgccttc
aggaaaccca cctttaccac 14640ctggctgttg tgactggcat cgctgaggct gtttctttgt
gtataaaatc gaataaggac 14700acatcacgtg ctagcaatcc accagcatag ggcgaaggct
cagcaaagag aagttctctc 14760ctcctctgct cttttaggtc agcagcaaat gcagatcggg
gtggggacaa ggtaaacaca 14820taacttgggt ggagatgtga ttgattaata actcataaat
catctgaaac catactttcc 14880ttttgatagt caaccctctg taaacactca atgtgttctc
accttgttat cattcccaat 14940gatgtcgaat gcgtggttgc cctctccagt ataaaagttt
gatgcagctt tgcctggatg 15000tacctgtcta taaggagtcc tgcttatcac aatggtaggt
aactggtttt atatataaag 15060gctagaaaag caaaagagaa tatgtattcc tattttaata
tgcgtgtgct tctaccacat 15120agcattgaat ggtaaatgtg acctagttag gaaccttatg
ttattacctg caattcaatg 15180tgcagagtat gagagtctga gagcttgcga aaattgtaat
gagtttagca ctagttctgt 15240tctctaagat ctaagttgtt tttccttcca gtaattctgg
ggttcaatta gatttcataa 15300gtgtctctaa ttcttgggtc ctatgtgagt aatttgaaat
taaaaataat ttcagagcta 15360catgactgca tcgtttttga gatgagggaa ctgagcctgg
ggaggggaag aagctgccac 15420tggggtctca ccgcaagcct gtgaatgagc tgagatagga
acccaggcat tcctctctcc 15480ctgccagggg ctcccaaact tggctgtgca gtggaaactc
cgggggaatt ttgaagaaca 15540ctgacaccca tcttccaccc cgacactctg atttaattgg
gctgcagtga gaacagagca 15600tcaatttaaa aagctgccca ggtcatttta atactcaagt
ttgggaacca tttccctagg 15660cccttttcac tgtatcatgt gttaaggaaa aatcttgggt
ttggggctgt gtttgttggt 15720ttatttgttt gtttttgaga caggatctca ctctgttgcc
caggctggag tgcagtggtg 15780cagtcatgat tcactgcagc ctggaccttc ctgggctcag
gtgatcctcc catctcagct 15840tcccaagtag ctgggaccac aggcgagtgc caccatgccc
gactaatttt tgtatttgtt 15900ttagagatag gattttgcta tgctgcccag gctggtcttg
aactcctggg ctcatgcaat 15960ctgcccacct cagcctccca aagtggtggg attactggtg
tgaaccactg tgcctggcca 16020agaaagaatt ttgcttcaaa atttataaaa acccatgcag
gcttttcttg tgtcttaaac 16080ttgatctttc ttaaaacatt ctttactaac gctggggact
gaactgggct gctctggggg 16140ctgcaaggga gatgaggcaa gaaacagcca gccaagtggg
aggagccaag ggaggtggga 16200gagggtcctc aggaggggac cccgggcctc ctctgggcca
ctcggcaaga caaacttgct 16260ctcagaaact gtccggaggc agaactgggg atggtaaaac
aacagtgggg tgggaggtcc 16320tccccatctc cactagacag gctcctccag cctatactct
atgcagcctc acagtgaact 16380tagcaagctg cagagctggc aatacctctg cctgcctaag
atcctgagga cccccaactc 16440tttatttatt tatttattta ttttattatt gttatttgta
gtagagatga gtttttacta 16500tgttgcccag gctggtctct aactcttggg cccaagtgat
cctcccacct tggcttccca 16560aagtgctggg attacaggtg tgagaaatta tgcctggcca
gacccccaac tcttgatcat 16620gggataaccc ttccaggagc agtaatttac ctaaagagat
acaacttttt tttttttttt 16680tttttttttt tttctgagac ggagtctctc tgtcgcccag
gctggagtgc agtggcgcaa 16740tctcggctca ctgcaagctc cacctcccgg gttcacgcca
ttctcctgcc tcagcctcct 16800gagtagctgg gactacaggc gcccgccacc acgcccagct
aattttttgt atttttagta 16860gagacggggt ttcaccatgt tagccaggat ggtctcgatc
tcctgacctc atgatccacc 16920cgcctcggcc tcccaaagtg ctgggattac aggcgtgagc
caccgcgccc agccaagaga 16980cacaacttct aaggatactg ccagcctgct tcacctcaag
gctcgctgta ccatctagaa 17040aatcctagcc cattctggcg tcgtggtgta atctcctgca
cacatcagga gctcagggtt 17100actaaatgca ggaaaggaag acacaaaggg aaggaaggct
ggtggcagaa tagaagacag 17160gaatgaaaaa agctttagag cctctctact caggggtgga
cctcagacca gcagcgtggg 17220catcacctgg gagcttccgt gtaagtctaa agacctactg
aagcagaatc tgcaggttaa 17280ttaacaggct gtctggttgg gcgcggtggc tcacgcccat
aatcccagca ctttgggagg 17340ccgaggcggg cggatcattt gaggtcaggt atttgagacc
agcctggcca acatgatgaa 17400actacgtctc tactacaaat acaaaaatta gccaggtgtg
gtggtgcact cctgtaatcc 17460cagctactca ggaggctgag gcaggagaat cacttgaacc
cgggaggcgg aggttgcagt 17520gagccaagat tgcatcactg aactctactc taggcgatag
aacgagattc cgtcccaaaa 17580agaaacaaac aaacagacaa acaaaacaaa acaaaaaaaa
ctcccaggaa tttatatgca 17640cattaaagca gcactgggtt ggtcattgtt cctgggttct
cacctgggag actaggtaaa 17700gacactgagg cactggctcc tagattgtgt ttcctatgag
ctccctggat aactctaatg 17760gtcacccaaa tgggagaacc cctcttttta cagggaagga
aaactgagtt tcagagagag 17820agagagagag agacagaaag ggagggaggg agggagggag
agagagagag agacagcatt 17880aggctatttc tgagatactg tcaagtggat aatctcaaaa
ttaaataatt tgctctctgc 17940tgtcaccatg tttgggctct cccccagtgg ttcaatctcc
tgcctctgct ctagagctgc 18000aggagacttg gggattttgc acatgtcact tgtcctggga
tgcagccctg cttgtccctg 18060ttgactctcc tgctttgtga ctcagtgaaa ccagatcccc
aagattccag tgatccccag 18120taccccagga gccctctttc tgcatgaagg gagtaaaaat
gctgtcaaca aggctggctt 18180tttgggggga aaagtttagg tttccacaat gatttcgagc
tgaaggctac tcagaagaat 18240gaaacagaac aagtgccact ccaagtcctg gcttgagctg
aatgtatcaa ggcgttgtgt 18300catttttaaa gaatctttct ttctttgtgg ctataaaact
catatatttt attgtagaaa 18360atttagaaaa taagaggaaa agtccaaaga agaaaattgt
cataatctcc ccatccaaag 18420atgacttgtt atttggtaca aaaccctcag tgtgttctga
gcatctctct ccgcatcatg 18480tatttcatag cctgcttcat ttcatgtcgt gacatgtggt
gatcaattgc tcatgtgact 18540tcaccttcta ccttgctggc tcaccaggtg tttttctgag
tcactgttct ccactggctc 18600tgttcttggg cattttgttt ctttccattt ttccactgca
gtgagcatcg ttgtggagga 18660gtctgtctct gtctctgtct ctgtgccatt tttaggatca
catcttggag atgaaaaggt 18720gaaatcaaat gttgtaaacg gttttaagaa tgtggatata
tagtagcaaa ttgttctcct 18780taacgattgt tcaaagtaag tcattcttgt gcatatcacc
tcccatagat acctctattt 18840ccctctgggt attataaatt aaatgattat tctcaccatc
ccattatgct tctatttcat 18900gctacatatt tacatttctt ttgttatcag taagttgaac
tttttcatgt gtttattcat 18960catttctgtg tgtgtgtgtc tgtgtgtgaa atgccacttt
atttctcttg ccctttttct 19020gtttaagatg tttttcttct cccattggta tgtaatgata
ttgttctgaa aatttgatat 19080ctttctaatt tcagttatgt tcttatgacg tttatattct
caaatctatt tttcttttat 19140gcttagaata agtatttatg tatatatgct gtgattattt
tatgatctta ttgtaaaaac 19200ttaaaccttg aatcctttta gaatttattt cactaatggg
gactcaaata ggaaattaac 19260tttttttctt ccccaaatag ttcaccactt gttctaatag
catttattga ataatcgctc 19320tccacagatc tcaaagaaca tgtttatttt ataataaact
tctacatata tttatacttt 19380ctgggttttg tttttttctt ttttacccta tccaactacc
tttggaattt tctgggtttt 19440ctattttgat ctctgtgttt gccttttttt ttttaaagac
agggtttcac tctgttgccc 19500aggctggagt gcagtggtgc tatcatggct cactgcagcc
tcaaccttcc tgggctcaag 19560tgattctcca cctcagcctc ccaagtagca gagactacag
ggacacacca ccatacccac 19620ctaacttttg tactttttgt agagatgggt tttcaccatg
ttgcctaggc tggtcttgaa 19680ctcctgagct caagcgatgt gcctgcctca gcttcccaaa
ttgctgagat tacagaattg 19740agacactgtg ccctccctat ctttctattc atatatgagt
aacatactac ttcaagtagc 19800gtcacttcat aattaatttt agcaatctta cagaataaat
gccttcattc ccattgtcaa 19860aagacaaaat tacaacaaat ttagtttaaa gatcttaatt
gatttttttt tttttttttt 19920ttttgagacg gagtctcact ctcttgccca ggctagagtg
cggtggcaca atcttggctc 19980actgcaacct ccgcctccca ggctcaagtg attctcccac
ctcagcctcc cgagtacctg 20040ggattactgg cgtgtgccgc cacgcccagc taattttttg
gtattttagt agagaatggg 20100tttcatcatg ttgcccaggg tggtttcaat tgcctgagtt
caggcaatcc acccacctcc 20160gcctcccaaa gtgctaggat tacaggcatg agccaccacg
cctggccctc aattaacttt 20220tacttgtgag tctataatca gggaacactt cattccataa
aatagatcga gtgttctgca 20280gagctgacca gagaagtttg gctttttttt tttttgagac
ggagtctcgc tctgtcaccc 20340aggctggagt gcagtggcca gatctcggct cactgcaagg
tccgcctcct gggttcgtgc 20400cattctcctg cctcagactc ccgagtagct gggactacag
gcacccacca ccacacccgg 20460ctaatttttt gtatttttag tagagacggg gtttcaccgt
gttagccagg atggtctcga 20520tctcctgacc tcatgatcca cccgccttgg cctcccagag
tgctgggatc acaggcgtga 20580gccaccgtgc ctggcagaga agtttggctt tatagacaga
aaagagccag aaaaagcaga 20640aacagagaac aaaaagcaaa ttggtctttt cgaagtgact
ttgcttataa gttaaagcag 20700aggggacttt ctcatcatgt cagctaaaac tggcctgttg
ggcatttggc tattacctct 20760cactctcttc taatttctta gaaggttggt taagcaactt
agttttggct tagtggcaga 20820gaacttcagc atgggcgact gcattttggt ttggtctgtt
gggcccagtg cagctcagtc 20880caaacaaatg gcctcctcaa catttttttt aacaccatct
tccctttcaa aagttcccag 20940aaattaccat gttaaggttt gtttgtttat ttagctcatc
tcaaaaagat gacaaagtca 21000actagatact tttccttgtt ctacaatttc agcctagatt
aactggtttg ttcagttatg 21060agctccacat cataacctct cagcagagcc cagccgagat
cctggtacac agcactcagt 21120caaggattat cctttgaatt aattcattct ccaccctgac
ctttcttcac attacagaat 21180gttctcctgg gcagcgttgt gatctttgcc accttcgtga
ctttatgcaa tgcatcatgc 21240tatttcatac ctaatgaggg agttccagga gattcaacca
ggagtaagtc ttggcttttc 21300aatgtttatt atgttattgc agcctggtag atggacctgt
ctgcagatga aagcctttgt 21360gtttctgttt gtttgtttct ttgttttttg agatagtcat
gctctgtctc ccaggctgga 21420gtgcagtggc accatttcag ctcactgcaa actcctcctc
ccgggttcca gtgattctac 21480ctcctcagcc tcccaagtag ctgggactac aggtgtgtgc
catcatgcct ggctaatttt 21540tgtatttttg gtaaagatgg ggctttgcca cgttggccag
gctggtctcg aactcctgac 21600ctcaggtcat ctgcccgcca cagcctccca aagtgttggg
attacaggtg tgagccactg 21660tgcctggcct cacttgggca gccttctaag aacacagtag
cctgggccct gtacccagag 21720atactgattt cattgttcta gggtaaggct gagacatggg
tggctttcaa gtacccaaga 21780tgtttcacat gtgcacccag ggttgaaaac caccgatgca
aagcaaaggg aagtgtagaa 21840gtgagattct catccagaga taatccagta ggaaaaaatg
tggtcccaag tacccacagg 21900ctcacatggc tatctaattc aataaaaggg ccttattttt
tcttgaaata tctgttctga 21960aataaaactt agaaggatat aagctttctg taagaaaaca
ttatcagtag catgaactga 22020acagtactgg aattagccgt gggtgaatac aggagcacca
ttcttagata cctttaaaat 22080aattgagctc ctctctctct gggaggggtg tagatagtag
gggcaggact caatgtcctg 22140ataggccttg ttctcattgc atgacttgca ctgatgatgt
tgtgtgtatt attttagaat 22200gcatggatct caaaggaaac aaacacccaa taaactcgga
gtggcagact gacaactgtg 22260agacatgcac ttgctacgaa acagaaattt catgttgcac
cctgtaagtc tcaagccagt 22320gctggccaag agggggacat agctgttctc tgaggaacag
ctactccgtc ttgcaaaatt 22380ccaatcttca ggggtttagt ttttgtttta gttttttata
gactttatat agagtagttt 22440tagattcaca gcaaaactga gcagaggtac agaaatttcc
catgtgcctc ctgccctcac 22500acatgcacga cctccccgtt atcaacatcg tccaccacag
tggtgtattt gttatgactg 22560atgaacctac cttgcacatc attatcacct gagtccatag
tttatgttag ggcttcctct 22620tggtgttgtg gtattggggg gttagttttc agtcctattt
ctttcttttt aaaaaattgt 22680gggccagtca caatggctca cgcctgtaat cccagcattt
tggggggccg aagcgggcgg 22740atcacgaggt caggagatcg agaccctcct agctaacacg
tgaaaccccg tctctactaa 22800aaataccaaa aaaattagcc gggcttggtg gcgggtgcct
gtagtcccag ctacttggga 22860ggctgaggca ggagaatggc gtgaacccgg gaggcagagc
ttgcagtgag ccgagatcgc 22920accactgcac tccagcctgg gcgacagagc gagactgtgt
ctaaaaaaaa aaaaaattgc 22980ggtaaaatat acgtaacgta aaatgtacca tgttaacaat
tttaaatgta tactttagtg 23040gcattaggtc cattcacatt gttgtggcat cactaccatc
cacctccaga acttgttcat 23100cctgcaaaac tgaagctcta tacccattaa gcagtcactc
tccatcccct tctccccagc 23160ccctggcaac caccatttca cttctgtctc tatgattttg
actactctag gtccctcata 23220taagtggaat catacagtaa ttgctttttt gagactggct
catttcactt agcataatat 23280cctcatggtt catctgtgtt gacgcttgca tcaacattgc
cttcttgttc gaggctgaat 23340catgttctgt aatatggaga gaccacattt tgcatctcca
ttcatctgtt gatgaacact 23400tgggttgttt tccacctcaa tctcatttct tttctgttgt
ttttgaaact tgataagtag 23460ctttattcca gatgggctcc tgaagacagg aaagagtatc
taattgtctg acactattcc 23520agagattggc ccggtcatca cctcagtctg ggccttgtag
atttgtagaa atagcagtgg 23580ctctgagcat aggcagaggg atcagatgtg aggggtaagg
gaggatctgt agtgtgcctt 23640gcaccataaa acttatgctg cttcactgct caagtttaac
tcacgtcagc ccaaaggcta 23700tgcagatgtt ttctgctggg ttgctatgga caccagaagt
ctagaacttg agcaggcgga 23760acacagagta cccgcagtgc cgtcctaatt tacctgtttc
ctcagcagct atgtccataa 23820cagctaccta actcccaacc ccccaaatcc atctaacctc
ttcatgtagc cttggcttgt 23880agtgttctgc aggaacagga atggatctga tggcagagca
ttggtgtcaa ggggcatttc 23940gcaaacttgt ctgatcacag actcacctgg gaagcttgat
gaaatacaga ttcctgggct 24000ccatcccaga actgattgat cagaatctgt aggggagggg
ccaaggaatc tgaattttaa 24060caggtgcctc gagagtctta tgaggacact gcgagtaaag
aagcaagcac ttgacttaga 24120gcagaacacc tgagtcctac tgaacttgaa gtgcctggag
agttccagcc acatttatcg 24180agcaggcctc atgctaggtg cctaatggaa cagaggaata
ttggactggg agatggctgg 24240ctgctcagag caggctcctg ggcatcgcat tggccagatc
actgggtatt acagctgaac 24300cttgattttg tggctccaga aatttgacat tgtctttata
tcaagtgttg atgtcactct 24360ggtggagtgt gattgtatcc cagccaggtg aaggactggt
gactggtccc tccctcacat 24420aggagcgtgg ctccctggtg gctaaaatgt ccatgtcacc
cagatttggc atccctgccc 24480tcttctaaga ttctccactg taggctggga ttctcgtggt
cctggtgtgg aaacgagcca 24540ggctgctcct tgcagtgaag agggaagaac ctcactgttg
gagtcagagt aacttgagtc 24600tgaatctcca ttcccttgta tactagctgt gtgaccctga
gtcagtttct gaatgtctct 24660gagcctctgt ttcctcacat tataattatg tggctatgat
aattaaatga gattctgtgt 24720atatattctt taacccaaca aacacttaat gccttctctg
tgccaattct gtgtcctgtg 24780atcctgggac agtaactgac acctggtcag gtttcaaggt
ttcaaggttt ctactgccat 24840gttgcctgtt ccatttggca cactgctcca tgcaggggaa
atgaatcaac accttcttcc 24900ctggaattgc ctcacctcct ttgtttttct catgaacttt
tttctcctcc atggaaaaat 24960acagactttg attaagcttt ttctggaaga agtttttgga
cccactgcac cctctatggg 25020acgctgccca gatctcaagg tgctcggtgg cagtggagcc
tgaggctggg ggcccaggca 25080gaggctccca ggtagggcat gggagatgtc ctgggctcca
cgctgcccgg gcccagggct 25140ggctccctga cttgggtgag gaaaagggag tgggagacaa
aaggccaagg tcatccagcc 25200actgactctg gttagctgaa tacccgatgt tgacagtttc
tccctccctc tctctctctg 25260ctgtttcctc tatagagcta agtctggttt gcattgaaat
aagcatgcag taatatgcct 25320tttgccttac caacttgtat ttgcacacag gcagcacaga
ttcaccattt cacttcctac 25380catgaactaa aagaattgta tctcttcaac aaacaccact
ttatttttaa cctttttttt 25440ttattgtggt aaaatgtata ctacataaaa tgtgccagtg
taaccatttt aaaacataca 25500attcagcggc attaagtaca ttcacagtac tatgcaacca
taaacactat ttccaaatct 25560ttttaatgac tccaatctga aactctatat ccattaaaca
ataactcaga ttccctgtcc 25620cccaaacctg tggtaatctc aagtctactt tctgtctcta
tgaatttgcc tactctagat 25680atttcacata aatacaatca cactgtgttt gtcttttcgt
gtctggctta tttcactcag 25740catgttgtca aggttcattc atgttgtagc atgtatcagt
acttcattct tgtttctgcc 25800agaataatat tccatggcat ggtcatacca cattttgctt
atccatttat ttgttgaaca 25860cttgagttgc ttctgcctgt tggataaagt aagtaatgtt
gttctgaaca ttggcataca 25920gtgtttgtca atatacaaac attggcatat aatgtttgag
tccctgcttt caattctttt 25980gggatatata cccaggagta gctttctggg tcatatggta
attttttgtt taacttattg 26040aggagctgcc aagctgtttt ccacagcagc tgcaccattt
tacattccta ccagccagca 26100atcaccttta aacatgaaaa acatgaaagg ttttaaatga
tatttcccaa ttttctaagt 26160aaacatatga attatagaat cattgggaag cataagagaa
aaaagaagaa atacctactc 26220ataatcccaa ctgtgtaaag gtaataacta ttaacattct
atttttattt atttatttat 26280ttgtattaaa aaaattaaga cagggtctca ctatgttgcc
caagctggtc tcaaactcct 26340gggatcaagc tgttgtctca cctcagcccc aagtagctgg
gattacaggc atgtgccacc 26400atgcctgggc ttctatcatc atttattttc tttttttttt
ttttgagatg gaatcttgct 26460ctgtcgccca ggatggagtg cagtggtgtg atctctgctc
actgcaagct ccgcctcctg 26520ggttcacgcc attctcctgt ctcagcctcc tgagtagctg
ggactacagg cgcctgcaac 26580cacgcccggc taattttttg tatttttagt ggagacgggg
tttcaccggt ttagccagga 26640tggtctcgat ctcctgacct cgtgatccgc ccgcctcggc
ctcccaaggt gctgggatta 26700caggcgtgag acacagcgcc cggccctatc atcatttctt
tatgtctctt ttctatatgt 26760tttttctttt acatagtgga tatcaaattt tatatcacag
ttttgtgggc tgatttttcc 26820acttaatatt ctagcactgg gctgggcatg gtgtctcacg
cctgtaatcc cagcactttg 26880ggaggacaag gtgggtggat catttgaggt caggagttcg
agacctgcct ggccaacatg 26940gtgagacctc ccccgcgccg acccgccatc tctactaaaa
atataaaaat tagctgggtg 27000tagtggtgtg ctcctgtaat cccagctact caagaggctg
aggcaggaga atcgcttaaa 27060cctgggagac ggaggctgaa gtgagctgag atcgtgccac
tgcactccat cctgggtgac 27120agagtgaaat tcatctcaaa aaattaaagt aataatagta
ataatacata aatattctag 27180cattagtatc atctcatctc atcagaactc ttcaaaatat
ttgtatgtac tgcagctcaa 27240ttagaggtgg tctctgactt tcatcagctt ctccctgggc
tccatgacac tggcctggag 27300tgactcattg ctctggttgg ttgagagagc tcctttgcca
acaggcctcc aagtcagggc 27360tgggatttgt ttcctttcca cattctagca acaatatgct
ggccacttcc tgaacaggga 27420gggtgggagg agccagcatg gaacaagctg ccactttcta
gagtagccag acttgcccct 27480gggcctgtca gcacctactg atgaccttct gtgcctgcag
gatggaatgt aggggtgagc 27540tgtgtgactc tatggtacag actggtccag gttcctggag
gtcacactct ggggctgcca 27600gccaaagggt gcccttaagt tggagtgtgg gtgacttcct
gaacagagct ggcactgggg 27660agggggtgct cggggtcctt tctcttctaa caggccccag
ttttgtctca atgacagtgt 27720ttctacacct gtgggttatg acaaagacaa ctgccaaaga
atcttcaaga aggaggactg 27780caagtatatc gtggtggaga agaaggaccc aaaaaagacc
tgttctgtca gtgaatggat 27840aatctaatgt gcttctagta ggcacagggc tcccaggcca
ggcctcattc tcctctggcc 27900tctaatagtc aatgattgtg tagccatgcc tatcagtaaa
aagatttttg agcaaacact 27960tgaatatgtg tgtcctttta atttataatt tatgtatgca
ttgatttaat acacattata 28020aagaatacaa aacatttaaa aagggagaca aaactacata
tgaatggccg ggcacagtgg 28080ctcatgccta taatcccagc actttgggag gccaaggcag
gcggatcatt tgaggtcagg 28140ggatcgagaa cagcctggcc aacatagtga aaccccgtct
ccactgaaat acaaaattag 28200cgtggtggca ggcacctgta gtcccagcta ctcgggaggc
tgaggcagga gaatcacttg 28260aacccgggag gcggaggttg cagtgagcca agatcacatc
agtgcactcc agcctgggtg 28320acagagtgag actccgtctc aaaaaacaag caaacaaaca
aacaaaccca aacccaaaca 28380aaacaaaaca aaacaaaata catatgaata aaagttctat
ttttcctaaa ctctataata 28440ggtcattgtg catcccttac tcaggggacc actgccactc
ttactgtttc tgatgtctgc 28500actctgctgc ccgtgtcctg ctgtctggtg aacgatgaaa
aaaatgtgtg tcttcaagct 28560ggaattagat cccttctttt ctttacggtg ttcctgttta
tccctccttc ctcttcatca 28620ggaagtgtct tctatgatgt gtcccccaca ttcctctgtg
tttgcttgag gtcaaagcag 28680cttagaacaa gaaacagctt agaacaagcc tgtaaaacag
cttagaacaa gcctgaagaa 28740agtgccccca agaccacatg cagcccgagg ggaattaaag
gccaggggct ctgccttgca 28800gatggagagt ggcctgggtg caaggcccca gcatcagcta
cctcttgcta cacacagacc 28860accgaaaact tagtggctta agacaacgac aacattgatt
ctcttttaac tgtttctgtg 28920ttaaagaaca tctgttcact caacaggtct gtactgagca
cctgggccag gcagtgctct 28980agacctgggg atacagcagt gaaccagaga gatttcagga
gagaaccctg ttctcctggg 29040gcttggactc agctgtgtgt acgtgacaga tcagaaatga
gcaaacagtc atctgttgaa 29100caatcagtgg gcaactgata aagagcttta ttttcccctg
tcccctatct gtttaaacat 29160gctatgtggg ccagttgagc aatctagaca ggtcccttag
ctttcttgtt caccttcaga 29220taatttaaaa aagtggtctg agttacattt tgagttacac
aaagtctcat tccaggggct 29280gcatctgaat tgagagggaa tgggggacag gagagttaag
ggctccttct gcctgaggtc 29340atgttctagg ctgacttcct catgggactt ttgtgggctt
ctctctccac agctcccctt 29400ccactcacag cctatgactg ttatcttagc ttctgcatcg
taatctatgt ctcctattgt 29460tttctaatga ggttgccttg ccccttgtag cagagtcact
cttggatgag atccataaaa 29520ccctgctatg atcacctcct ttgctgcgga atgtggggaa
gcagttggca actgagtatg 29580gaggacctct gtcaaaactg agacagaaag aagtgctatt
taacatctag tcacaagtaa 29640ataatgtggg ttataatctg ggctgtaggc tggacgtggt
ggctcatgct tgtaatccta 29700gatcaggtgg gaggatcgct tgtgcccagg actttgagac
cagtctgggt aacacagcaa 29760gaccctgtct ctacaaaaaa atttaaaaac cgggcatcat
ggcgcatgtc tgtaatccca 29820gctactcagc aggttgaggc aggagaatcg cttgaacaca
ggaggcagag gttacagcaa 29880gccgagattg cgccactgcg cctgagcgac agagtcagac
cctgtctcaa agaaaaaaga 29940aaagaaaaaa agaatctggg ttctgccatt tatttattta
ctcaattttt tttttttttt 30000630000DNAHomo sapiens 6ttttaaaatt aaaaataatg
tttgttagga gttataaatt atgattaatt aattatgggg 60aattataaaa tatgattgta
tgagattttg atggtttata aagtgtattt attgttaatt 120attttaaata ttaatgaatt
taaaaatgaa tttacggaga ttggaatgtt ttttttttgt 180tgtattagtt ggtttaggtt
gttataataa aatattatag attgggaggt ttaagtaata 240gaaatttatt ttttatagtt
ttgggggttg gaagtttacg attaaggtgt aggaaaggta 300ggttttattt tgaggttttt
tttttggttt atatgtggtt atttttttat tgcgtgttta 360tatgattttt ttgtgttttt
ggaaagaggg tgtgggggat agagggaaag agaaggagag 420ggaatttttt ggtgtttcgt
tttttaagga ttttaatttg ggttattttg gtttaggtat 480tgtggggtgg ggggttgtgg
ttgttttgtt ttgagtggtt aagataaagt aatagaaaaa 540tgtttaaagt tgtgtagtaa
agataagtta tcgaataggg atttgtttat tagtgtgggg 600atttttaagt cggttatttt
ggaggtaagt ttttatagag tttatgtaag gtggtagtag 660tagaagaagg gaattgtttt
tgtttttggt atatttttta tcgatttggt gatgttggat 720attgcgatga atggtaatgt
ggatgagaat atgatggatt tttagaaaag gagatttagt 780tgtttaggtg gttgtaaatt
attatagttt ttattttggg gaggaattgg gggtttggtt 840ttgggttaga gagtagttta
gtgagggtga gagttatagt ttgttttgtt agttggattt 900ttagtttcgg ttaattagta
attaaggttg agtagattag gtttttcgga gttggttttg 960ggaagttagt tttggggtga
gttggttttt gttgtggtat tgagataata ttgttataaa 1020tttaatgcgt ttttgtattt
tttttttttt tttatttgtt tatatttata attattatgt 1080atattagttt ttgttagtgt
ttttatttaa tttaatagag atatgttata tttaaaaaaa 1140aaaaaaaaat gggtcgggcg
tagtggttta tgtttgtaat tttagtattt tgggaggtcg 1200aggtgggtgg attatgaggt
taggagatcg agattatttt gattaacgtg gtgaaatttc 1260gtttttatta aaaatataaa
aaaaattagt cgggcgtggt ggtaggcgtt tgtagtttta 1320gttatttagg aggttgaggt
aggagaatgg cgtgaattcg ggaggtagag tttgtagtga 1380gtcgagatgg cgttattgta
ttttagtttg ggtttagttt gggtaataag agtaaaattt 1440tgttttaaaa aaaaaaaaaa
aaagataaat aataaaaaat aaaatataaa taaaaaagta 1500aattgtagat ttttagtata
aatgatttta tttatttttg gtattaagag attttaataa 1560atattgttgt ttttaatagt
ttatggttat atttttagga tgatgatttt agttttttta 1620agtttttgtt taataaaatt
taatgttgtt taaagaattt atgatttgtt ttaggtaaaa 1680tttgaatata ggttttcgaa
tttttttttt ttaaaggatt tattttagaa aattggtagt 1740tagaaatttt ttttttgttt
tttggaaatg tattttttat aatataggaa tgtttttttt 1800aaggatttgg gagttatttt
ttcgaaatgt aattattagg taggatagag tttttgtttt 1860ttaattttgg tggtagggta
ggattttgat attgataagt attagtttat aatttgataa 1920gtataagttt ataatttggt
tagtgtattt attgattaaa tttttttttt tttaattagt 1980attttttagt attttttttt
taatatattt tagtatttaa aaagtttttt gttttttggt 2040ttggtagaat tgagtttagt
taatgttgaa gtgttttttt tttgttatag tagtttgaat 2100aaaatttgtt ttgatatttt
tgataagtgt ttggtgtaaa attttttttt aataatagta 2160agtgttgaaa ttggatagtg
taagtttttt ttttagtttt gttttttttt agaatattta 2220ggttatttta ggttattcgt
ttttttatat aaattttagt aaaagtttat taatttttat 2280aaaaatttgt tggattattt
tttttgtttt tgtttttgag attaagtttt attttgttat 2340ttaggttgga gtgtggtggt
ataattttgg tttattgtaa ttttcgtttt ttaagtttaa 2400gcgatttttt tgttttagtt
tttttagtag ttgggattat aggtagatgt tattatgttt 2460agataatttt tgtattttta
gtagagttat tgttttatta tgttggttag gttggttttg 2520aatatttgaa tttaggtgat
ttatttgttt cggtttttta agtgttggga ttataggcgt 2580gagttattgt atttagttag
gattgatttt ttaatttttt tttttgattt cgtagtattt 2640ggaatggatt tattttttta
ggaacgatat taattttttt taaattggta gaaatttatt 2700aataaattgg gggaggattg
ttattttaat aatagagttt tttaatttat gaatatggga 2760ttatttaaat tagggattag
taaatttttt gtaaagaatt agatagtaaa aatatttgtg 2820gtattgtggg ttatatttaa
ttttgttttg tagtgtaaaa gtagtgttgg ttcggcgcgg 2880tggtttatgt ttgtaatttt
agtattttgg gaggttgagg cgggtggatt atttaaggtt 2940aggagtttaa gattagttgg
ttaatatgga gaaatttcgt tattattaaa aatataaaaa 3000aaatagttag gtatagtggc
gtacgtttgt aattttaatt attcgggagg ttgagatagg 3060agaatcgttt gaatttggga
ggtggatgtt gtagtgagtt gagattatat tattgtattt 3120tagtttgggt aataagagta
aaattttatt ttaaaaaaaa aaaaattgtg ttttaataaa 3180attttattta taaaataaat
agggggttag tttagggtta tggattgtag tttgttgttt 3240ttaatttaga atttttttaa
tttttttgag taaagtttta tagtttttat tgtaagtttt 3300gtattttttt tattaaattt
atttttaagt attttatttt tttaggtgtt attgtgaatg 3360aaattttgtt tttttaattt
tatttttata ttgtttatta ttagtatgta gaaatataat 3420tgatttttat atatcggttt
tatatatttg aattttgttt gttaaattta tttattaatt 3480ttagtaaaat tttttgatag
attatttggg attttttgta tatagattat gtggtttata 3540aataaatata gttttatttt
ttttttttta ttttagatgt ttttaattta tttttttttt 3600tttttttttt tttatttttt
ttttaagttg ttttggttat gattagtagt ataatgttga 3660atggaagtgg taagagtaga
tatttttgtt tgttttttaa ttttgagtgg gaaatatttt 3720gttttttatt attaagtata
ttaattgtag gttgtttgta gttgtttttt attagattga 3780agaagtttat ttttattttt
agtttgttga gagtttttat tacgaatgta tgttggttat 3840tgttaaatgt ttttgtattt
attgaaatga taatgtgatg tattttagtt tattaattta 3900gtatattata tttattgatt
ttgaggtttt gttgttttta attaattaat tttaaaaatt 3960ttattatata ttagagaaag
tattatttga tttttggatg ttatattaat tttgtatttt 4020tgggataaat tatatttggt
tatggtgtat aattttttgt atatgttgat ggatttttgt 4080ttgttaatat tttattgagt
atgtttgtat ttttatttat gagagatatt gttttataat 4140ttttgtttat tttggtgtgt
ttgtttagtt ttgatattgg ggtaatattg gttttattaa 4200aggagttgga atatgattta
ttttttttta ttttttgaaa gatttttgtg aaagatataa 4260tatttttttt cgttttattg
tgtcgtgtcg tgttttgttt tgtttttttg aggtggagtt 4320ttgtttttgt tgtttaggcg
ggagtgtaat ggtatgattt cggtttattg taatttttat 4380tttttaggtt taggtgatgt
ttttgttttt taagtagttg ggattatagg tatgtattat 4440tagatttggt taattttttt
tttttttttt tttttgagat aaagttttgt tttgttattt 4500aggcgggagt gtagtaacgt
gattttggtt tattgtaagt ttcgtttttc gggtttacgt 4560tattttttta ttttagtttt
ttgaatagtt gggattatag gtattcgcga ttatatttaa 4620ttttttttgt atttttagta
gagacggggt tttattacgt tagttaggat gatttcgatt 4680ttttgatttt atgatttatt
cgtttcggtt ttttaacgtg ttgggattat agttatgagt 4740tattatgttt tgtttaattt
tgtattttta gtagtgatgg ggttttatta tgttgattag 4800gttggttttg aatttttgat
tttaggttat ttattcgttt ttgtttttaa tttggttaat 4860ttttttattt tgtagagatg
gagtttttat tgtgttgttt aggttgtttt tgaatttttg 4920gttttaagta atttttttat
ttgggttttt taaagttttg ggattatagg tataagttat 4980tatttttggt ttaaaaatgt
attttttata tatatgtttt tgttaaaatt gggataaaga 5040gaaatagatt ggtagtggta
tgtattttaa ggaaaatagg agaaagttta tttatttagg 5100aaagtgaggt ttataagtat
tttatatata aatattatat ttgttttatt gggttgtgtt 5160atttgatttg tttcgagtat
ttgaatttgt tatttttaat agagaattga tttttttatt 5220agttaaagag gaatttgaga
tttggtgagg gtgagtattt ggttagttat tagtaagttt 5280ttttttattt tatttatcgg
cggtgattag taattatgtt tgataggtat ttattttttt 5340tttggtgagt agttggaaaa
gagagtagtt tggaaatatt tggaaatagt atcgtttgtt 5400ttttatatat gtgatttgga
agttttttgt ttgttttata ttgttttgtt tttttagatt 5460gaattagtgt ttattttata
tatgttgagt gatggtttat gtttttttaa aaggtataaa 5520attaagttgt gttttgatta
ttttgggtat atgttgttag aattttttga gggtgtgtta 5580cggtagtata tttttaattt
tggcgatttg gtgttaacgt tgtattaaat tattaggttg 5640gttaatattt attggaattt
tttggtgtta aaggttgtgg gagatttgaa tatattgtta 5700agggaggagg aaggggaagg
agaagggttg tgggattttt cgtatgaaag gttttatgtt 5760ggtggtagtt tttatttgtt
gttttggggt ttaaaggtgt ttagattttt ttatttgagt 5820gtagatgttg ttagtttttt
gggtaggtgt gtgagggagg tagttttggt tgtaaagaga 5880gtaggggttt ggttgggcgt
ggtggtttac gtttgtaatt ttagtatttt gggaggtcga 5940ggtggtagat tatttgaggt
taggagtttg tgattagttt ggttaatatg gtgaaatttt 6000atttttatta aaaaaaaaat
atagaaatta gttgggtatg gtggtatgta tttgtaattt 6060tagttacgta ggaggttgag
gtaggagaat tatttgaatt taggaggcgg aggttgtagt 6120gagttgagat agcgttattg
tattttagtt cgggcgatag cgcgagattt tattttaaaa 6180aaaaaaaaaa aaaaaaaaaa
aagagagtat tggtttttgg gttattagga ttattatata 6240attgaattgt gttaggtttt
tgagttgaag tttagttatt ataatttttg tgatttgtat 6300atatatgttt agatggtttg
taggaattaa gaagtttgga gtagttaaaa aatttataaa 6360gtaaaatagt tagtttttgt
tttaattgag taattaaaat tataatattt tattattgtg 6420atttgttttt gttttatttt
agttgattaa ttaattttgt gatatttttt ttttggataa 6480tgagttttat gattttttta
ttatgtattt tgtgattttt ttttttgtta ataatagata 6540attacgtttt attgtaattt
tttattattt atttaatttt tataaagtaa tttttttttt 6600attttttttt gttgattttt
ttcggattta gtttatttgt atttaagtga attaaaagtt 6660ttatttttta tataaagttt
gtttggtggt ttttttatat ggacgtgttt gataaattgg 6720ggtcggtttg tttagtgtag
ttaagtttag atatttatat tgaagttgta gtattagaaa 6780ggaaggtgtt tatttataga
gttttgagta agggggatta ggtagcgtat gtttaaaatt 6840tgaatttttt gatggttggt
agatgtgaat tttgaaaatt tgagataagt gttaatttag 6900aaagtttatt ttgttaaggt
tgaggatatg ttttcgtgat atatttttag gaggttttga 6960cgatatgtgt ttaaggtggt
tagagtatag tttggtttta tattttttag ggagatatga 7020gttattaatt aatatatgta
aaatgaatat tggtttggtt tggaaaggta ggataatttg 7080aagtaggtag tgggttttta
gattatagat aggtgagaga taaacggttg tatttttttg 7140agtttttgat tagttttttt
aaaggaggta attagatatg tatttatttt agtgagtaga 7200gggatgattt tgaatagaat
gcgaggtaga tttgttttaa gtagttttta gtttgaattt 7260tttttttaat ttagtgattt
gggagggtta atatagtttt tttttatata ggtaagggtt 7320tttaaaggcg gggtaaattt
ttaggaaagt agtagaagtt agataaaatt gtaaattagt 7380atatgaaggg tatatattgg
tttggtttaa aaaaggtgag atattttgaa gtaggggttt 7440ataggttata ggtggattta
gagatttttt gatttgtagt tggtttagga ggcgatgttt 7500tgtttgaaaa tttagggtta
atagaaaaga atattagttt tagtttatgg gtgtggtttt 7560taggagtttt cggaagaaat
ttagaatgaa agaaggtggt tagagtgtag tttttagttt 7620tttttatttg aggtttgtag
gttagtagat tcgtttggtg ggggtttata tggaggttta 7680ggtttttgaa tgataaattt
aaggacgtgt gttaagatgt tatttttagt ttttaaaatt 7740aaatattttc ggattttaat
ttttttggtt attgttttgg gttattatta tttttttgtt 7800taataagtta tttatttatt
ttttagggtt agttggatgt ttgaaatttt ttttgaaggt 7860atttaagatt tttttttatt
tttatgtttg atggtggcgt tcggggtggt ggtataggtt 7920tttgttttat tttaggtttg
ggtttaaatt tttatttttt tttattattt gaaaattttg 7980aatagttgtt tttattttga
aattttaatg ttttattggt aaagtgggcg agagaaggtt 8040tgttttgttg tgttgttggg
aaatttttga ggtaatgtat gtgtggatat gagaggtttt 8100agtttttttt ttatttttta
gttttttatt ttggggtggt tttggaaggt attgtttatg 8160gtaaggatag aaggttgttg
gttggcgttt ttttgtttga tttagtggtt gtatttattt 8220agtatgtgtt ttatgaggag
aataagggtt tgtgatgttg gtatagatta gtttagatat 8280attggtgggt acgtggttgt
tgtttaatat ttttgagtgt ttttatttag tatgttggat 8340tttggttttt ttagatttta
atattattta gaaatagaag gaatagagtt tcgtatagta 8400gggtttttta gattgatttt
tggtagttga attgaagggt tagtggttga gttatattgg 8460ttgtttaata ttttgaatat
tatatttaga aagaaaatat aagttttttg ttacggtagt 8520ttttttgtga ttgtgttata
tttaatataa gataaatatt agttttataa aagggagtag 8580tttttgggat aatttattga
aagcgtgaga ggtaggtagt ttttgggttt tgtgtatagt 8640tgttttaagt tgtaaatata
taatttttga ttaaatattt tttaatttat ttgataaata 8700agaagttttc gggtttttaa
ggggatgttt aagttttttt tttttttggg ataaaatttg 8760gttttgtttt ttaggttttt
tagcggttaa tttggaatat gtttgttttt gtttcgtttt 8820tttgttttag tgtttttttt
gttgttttta tatttttttg gataagtttt ttaagttttt 8880tatagaaaat tagaaagatt
atttattagt agtttttttg gtttttggtt tgttagtttt 8940aatgtatttt agtgtatttt
tgtgatattg tatttagagt agttgtggtt tatagtattt 9000atagtgtttt ttttcgttat
tttattagtg gttttttttt ttaatttagt tttttgtttg 9060gatatttagg atttttattt
tatcgttatt tttattttat atagggggtt tattttatag 9120tatttttgtt gttgtaaaaa
gattttttat tgtatttttt ggtatggaga atttgtttta 9180ttgtattttt gaagtaagag
attatgtaga aatgaattta tgggtggggg tagagtttgt 9240ttttttggtt tttgtagagt
gaaaattttt ataatgtttg ttttagatta agtttcgtag 9300tttagaagtt ggtttatttt
tggatttaat atttgagttg aaaacgtttt tttttgttta 9360tgtttatttt agttcggttt
ttaatagaat agaattttga cgaatattaa aacgggtttt 9420gttatagggt attataaaga
ttgggtttta taaagtaaaa ttgttagggt ataagtggat 9480ttttagggag gttggtttat
gttaggttgg gaattgggta gttttcgaat attgaatatt 9540tttttatttg tttttttatg
tttgtttgtt ttggggaggt tggtttttat ggaggggttt 9600ttgggaagga atgggggtcg
atggggaggg gaatgggatt cgtattgcgt atttggggtg 9660aggaagggat ttagagggaa
agtggaaaga ataggagtcg gtggagggta atgtgtaggg 9720taggggtggg ggtgtagttt
ttatttttgg tttttgtgag atacgaaata agattgtgtt 9780ttgcgtatga agtaggtagt
tgggtgagag gtttaagaga agagattatg gtttggaaag 9840gttttagagt acgatgggag
aggaagtgga tgagagataa gttattagtt tgaagattgg 9900taaggggaga ttttatcgat
tttggtgtta aattaagttt agtttaaagt ttttttttta 9960tatattttaa atttatttta
atggtttttt tgtatatagt gaattgtaag ttaattgtac 10020gtgtaaatag gttgttattt
attttcgtat taagtagtcg agttttagtt aattatagta 10080gttagatttt aattatttat
aggcggttag ttgttgaaat tggatttaaa taagttaagt 10140attatgttat tattaattcg
tttgtttttg tattttattt ttagtttttg tatattattt 10200tttttttttt gtttataaat
ttttgattat gaggtagtgt aggagttttt ttgaatttat 10260ttttgtttgg gggttgtttg
atttatataa atggtttttt gtttaattaa attttgttaa 10320atttaatttg tttaacgttt
ttttaatatt gggaatgatg gtagggtacg gtggtttatg 10380tttgtaattt tagtattttg
ggagattaag gtaggtagat tatttgaggt taggcgttta 10440agattaattt gggtaatggt
gaaattttgt ttttattaaa aatataaaaa ttagttaggt 10500atggtggtgt atgtttatag
ttttagttat tgggaaggtt gaagtataag aatcgtttgt 10560gttcgggagg tggaggttgt
agtgagttga gattgtagta ttgtatttta ttttgggcga 10620tagagtaaga ttttgtttta
gaaaaaaata aaaaataaaa taaaataaaa aattttggaa 10680atgaatggta gtttttgtta
tagtattagt ttaagggttg tgtgatttta tggattttga 10740ggtgtttttt ggggtttgta
gtgttgattt ataggtgggg atagtgtggt tgtagttagt 10800taaagtttaa aatttttata
tgttaaaata tattatttga gaggtaaaaa tataaatgag 10860gaaaattttt tattttttag
ttttaatttt agaatgggtg tgttgattat gaaaaaaatt 10920agggaaatat tttaatatgt
gtgatatttg aagattaata tttttaatat tagaagtttt 10980aattaattta gaagaaaaat
ataaataatt taaatagaaa aatagttaaa ggatatgagt 11040gaatagggta tttataaaag
aagaaaaata attggttaat atatatataa aaaaaggttt 11100aggttgtgta cggtggttta
cgtttgtaat tttagtattt tggaagttcg aggggggtag 11160attacgaggt taggagatta
agattatttt gattaatatg gtgaaatttc gtttttatta 11220aaaatataaa aaattagtcg
ggtgtgatgg agggtgtttg tagttttagt tattcgggag 11280gttgaggtag gagaagggcg
tgaatttgga aggtggagtt tgtattgagt tgaaattgta 11340ttattgtatt gtagtttggg
agatagagcg agatttcgtt ttaaaaaaaa aaaaaaaaaa 11400aaaaaaaaag gtttaatttt
gttaattagt aaagaaaaat aaattaagat aaagtatttt 11460ttatatatta tagtaatagg
tgataattta agagaattat aatttttttt tatttagaaa 11520ttttattttt gtgatttttt
ttttttttga gatggagttt cgtttttgtt gtttaggttg 11580gagtgtaatg gcgtgatttt
agtttatagt aattttcgtt tttcggtttt aagtgatttt 11640tttgttttat ttttcggagt
agttgggatt ataggtatgt tttattacgt tcggttaatt 11700ttgtattttt agtagagacg
gggttttttt atgttggtta ggttggtttc gaattcgtga 11760ttttacgtga tttatttatt
tcgttttttt aaagtgttgg gattataggt gcgagttatt 11820ttattcggtt tttatgaatt
tattttaaag aaattagtat aagtgtggga aatttttatt 11880tgaaggattt ggattttaat
agcgtttttt tttttttttt ttgagataga gtttcgtttt 11940ttcgtttagg ttggagtgta
gtggcgcgat tttcgtttat tgtaagtttc gtttttcggg 12000tttacgttat ttttttgcgt
tagtttttcg agtagttggg attataggcg ttcgttatga 12060cgttcggtta atttttttgt
atttttagta gagacggggt tttattatgt tagttaggat 12120ggtttcgatt ttttgatttc
gtgattcgtt cgtttcggtt ttttaaagtg ttgggattat 12180aggtgtgagt tattatgttt
agtttagtgt ttttaattat agaaaaaaaa gttagaaata 12240atttgagtgt ttaataagag
gtatttgatt aaataaatga tggtttatat tattttatgg 12300aataaaatgt agttattaaa
atgataatta gtttgggtat ggtggtttat gtttgtaatt 12360ttaatatttt gggaggagga
ggtaggagta ttatttgagt ttaggagttt aagattaggt 12420tgggtagtat agtaagattt
tgtttttata aaaaaaattt aaaaattagt tgggtgtggt 12480ggtacgtttt tgtagtttta
tttatttggg aggtttaggt gggaggatta attgaattta 12540ggaattggag gtaatagtga
gttataatgg tgttattgta ttttagtttg agtagtagag 12600taagattatg tttttaaaaa
aaattaaaat aaataaaaag aaaaataaat aattaaaaga 12660aattaagttg gttttattta
taaggatgat agttgaggta ggattaatat tgaaaattga 12720ttaggtaggt ttggatagag
ttggttgtta tatattcgga gagtattgtt tattttattt 12780gttttttttt ttgtgggtag
ttggttatat ttgttttgag tatttttttt ttaatttttg 12840gggagtagta gatgagttta
ttttagtagt gatatgggtt ggttttgtta ggatattttt 12900tatttgggaa tatttgagat
aatttaggtg aaattttgtt ttgggtagtt gttttgtatt 12960aggtattttg ttggttattg
gaaggtatat agataggaat aggttatagt tattgtttgg 13020gttataaagt ttttaattgt
aaataataga atttatttta ggttatttga ggtggaaaga 13080gatttgttag agaatttaag
tttttattaa cggttagaga tataggtttt tttggtataa 13140gtttaggtag agtattgaaa
ttatattgtt gtttggtttt agtaagatta ttttgttatt 13200agttatagtt atagttttta
tgtattgata ttgtatattg gattttaaga aatttttgtt 13260ttaagagtta gaatttttta
tattattatt tttagggaga ttttgtatga tgttagtgtt 13320tttggatttt ttttgaagag
gattttttga gaggagagtg tttgtggagt cgaggtttgt 13380gtttgtattt tggaggtaag
taggtgaggg tagtaaattt tagttttttt taatgtaagg 13440tagaattttt tattttatat
tgtgggaagt tttttaaatt tggagtttta aaagaattta 13500aggggttata gtgtatgatg
aatgtgtttc gattttttgg aatttagagt cgatgaatat 13560ttaatttatg taggtaaagg
tagaagtgtg tggtatcgtt ggtgagtgag gaggtagtta 13620gatagtatta tagttttttt
aaggagtagg agagtgattt taagttgtgt taggggtggt 13680gaggttgatt ttagggaggg
tagaggatgg tgttggaaag ggttattgtg gttagtttag 13740atgagtttta aggatttttt
ggttaggagt atgaattttt atttgtgggt agagggattt 13800taggttaaat tttaggaatt
gtagtaagtt gtggcgagga gtgaggggat tttttgggat 13860aaaattgttt tatgagttta
attgtatttt ttaaaaattt attggtttaa gttttaattt 13920ttaatatttt agaatgtgat
tgtaatggag atacggtttt taaagaggtg gtttaggtaa 13980aatgagatag ttagagtgga
ttttaattta atatagttag tgtttttatt agaagaggaa 14040atttggatat agagagagat
gttagggatg tatgttatgt aagaggtagt aagagtatgg 14100ttatttgtaa attaaagaga
gagggtttag aggaaaataa gtttttttta atggtttgat 14160tttatatttt tagtttttag
aattgtgagt aaataaattt ttttagttaa agttatttag 14220ttgtagtgtt ttgttgtggt
ggtagagtaa atagataaag tagtaagttt ttttagtatt 14280ttaggaggtt ttgtttgttg
ttatttttta aaggttattt tagttatgat gagggtaggt 14340gcggttgttt gggaggagat
tattgtggaa taaagagaaa taaggtttgt ttgatgtagt 14400agggagtaga ttatagattt
aagaagggtt ttagaaaggt ttagtaattt agtttgttag 14460ggaatttcga gttgtatgta
gtttttgttt ttttgtgttt tgtaggatgg tttgatttat 14520ttttgttttt atgttagatt
ttaagggtag ggttatttgg gttatagatt taggttcggt 14580gggtttggtg ggttgatttg
tgggttattt ttatgttttt aggaaattta tttttattat 14640ttggttgttg tgattggtat
cgttgaggtt gtttttttgt gtataaaatc gaataaggat 14700atattacgtg ttagtaattt
attagtatag ggcgaaggtt tagtaaagag aagttttttt 14760tttttttgtt tttttaggtt
agtagtaaat gtagatcggg gtggggataa ggtaaatata 14820taatttgggt ggagatgtga
ttgattaata atttataaat tatttgaaat tatatttttt 14880ttttgatagt taattttttg
taaatattta atgtgttttt attttgttat tatttttaat 14940gatgtcgaat gcgtggttgt
tttttttagt ataaaagttt gatgtagttt tgtttggatg 15000tatttgttta taaggagttt
tgtttattat aatggtaggt aattggtttt atatataaag 15060gttagaaaag taaaagagaa
tatgtatttt tattttaata tgcgtgtgtt tttattatat 15120agtattgaat ggtaaatgtg
atttagttag gaattttatg ttattatttg taatttaatg 15180tgtagagtat gagagtttga
gagtttgcga aaattgtaat gagtttagta ttagttttgt 15240tttttaagat ttaagttgtt
ttttttttta gtaattttgg ggtttaatta gattttataa 15300gtgtttttaa tttttgggtt
ttatgtgagt aatttgaaat taaaaataat tttagagtta 15360tatgattgta tcgtttttga
gatgagggaa ttgagtttgg ggaggggaag aagttgttat 15420tggggtttta tcgtaagttt
gtgaatgagt tgagatagga atttaggtat tttttttttt 15480ttgttagggg tttttaaatt
tggttgtgta gtggaaattt cgggggaatt ttgaagaata 15540ttgatattta ttttttattt
cgatattttg atttaattgg gttgtagtga gaatagagta 15600ttaatttaaa aagttgttta
ggttatttta atatttaagt ttgggaatta tttttttagg 15660ttttttttat tgtattatgt
gttaaggaaa aattttgggt ttggggttgt gtttgttggt 15720ttatttgttt gtttttgaga
taggatttta ttttgttgtt taggttggag tgtagtggtg 15780tagttatgat ttattgtagt
ttggattttt ttgggtttag gtgatttttt tattttagtt 15840ttttaagtag ttgggattat
aggcgagtgt tattatgttc gattaatttt tgtatttgtt 15900ttagagatag gattttgtta
tgttgtttag gttggttttg aatttttggg tttatgtaat 15960ttgtttattt tagtttttta
aagtggtggg attattggtg tgaattattg tgtttggtta 16020agaaagaatt ttgttttaaa
atttataaaa atttatgtag gttttttttg tgttttaaat 16080ttgatttttt ttaaaatatt
ttttattaac gttggggatt gaattgggtt gttttggggg 16140ttgtaaggga gatgaggtaa
gaaatagtta gttaagtggg aggagttaag ggaggtggga 16200gagggttttt aggaggggat
ttcgggtttt ttttgggtta ttcggtaaga taaatttgtt 16260tttagaaatt gttcggaggt
agaattgggg atggtaaaat aatagtgggg tgggaggttt 16320tttttatttt tattagatag
gtttttttag tttatatttt atgtagtttt atagtgaatt 16380tagtaagttg tagagttggt
aatatttttg tttgtttaag attttgagga tttttaattt 16440tttatttatt tatttattta
ttttattatt gttatttgta gtagagatga gtttttatta 16500tgttgtttag gttggttttt
aatttttggg tttaagtgat ttttttattt tggtttttta 16560aagtgttggg attataggtg
tgagaaatta tgtttggtta gatttttaat ttttgattat 16620gggataattt ttttaggagt
agtaatttat ttaaagagat ataatttttt tttttttttt 16680tttttttttt tttttgagac
ggagtttttt tgtcgtttag gttggagtgt agtggcgtaa 16740tttcggttta ttgtaagttt
tatttttcgg gtttacgtta tttttttgtt ttagtttttt 16800gagtagttgg gattataggc
gttcgttatt acgtttagtt aattttttgt atttttagta 16860gagacggggt tttattatgt
tagttaggat ggtttcgatt ttttgatttt atgatttatt 16920cgtttcggtt ttttaaagtg
ttgggattat aggcgtgagt tatcgcgttt agttaagaga 16980tataattttt aaggatattg
ttagtttgtt ttattttaag gttcgttgta ttatttagaa 17040aattttagtt tattttggcg
tcgtggtgta attttttgta tatattagga gtttagggtt 17100attaaatgta ggaaaggaag
atataaaggg aaggaaggtt ggtggtagaa tagaagatag 17160gaatgaaaaa agttttagag
tttttttatt taggggtgga ttttagatta gtagcgtggg 17220tattatttgg gagttttcgt
gtaagtttaa agatttattg aagtagaatt tgtaggttaa 17280ttaataggtt gtttggttgg
gcgcggtggt ttacgtttat aattttagta ttttgggagg 17340tcgaggcggg cggattattt
gaggttaggt atttgagatt agtttggtta atatgatgaa 17400attacgtttt tattataaat
ataaaaatta gttaggtgtg gtggtgtatt tttgtaattt 17460tagttattta ggaggttgag
gtaggagaat tatttgaatt cgggaggcgg aggttgtagt 17520gagttaagat tgtattattg
aattttattt taggcgatag aacgagattt cgttttaaaa 17580agaaataaat aaatagataa
ataaaataaa ataaaaaaaa tttttaggaa tttatatgta 17640tattaaagta gtattgggtt
ggttattgtt tttgggtttt tatttgggag attaggtaaa 17700gatattgagg tattggtttt
tagattgtgt tttttatgag ttttttggat aattttaatg 17760gttatttaaa tgggagaatt
ttttttttta tagggaagga aaattgagtt ttagagagag 17820agagagagag agatagaaag
ggagggaggg agggagggag agagagagag agatagtatt 17880aggttatttt tgagatattg
ttaagtggat aattttaaaa ttaaataatt tgttttttgt 17940tgttattatg tttgggtttt
tttttagtgg tttaattttt tgtttttgtt ttagagttgt 18000aggagatttg gggattttgt
atatgttatt tgttttggga tgtagttttg tttgtttttg 18060ttgatttttt tgttttgtga
tttagtgaaa ttagattttt aagattttag tgatttttag 18120tattttagga gttttttttt
tgtatgaagg gagtaaaaat gttgttaata aggttggttt 18180tttgggggga aaagtttagg
tttttataat gatttcgagt tgaaggttat ttagaagaat 18240gaaatagaat aagtgttatt
ttaagttttg gtttgagttg aatgtattaa ggcgttgtgt 18300tatttttaaa gaattttttt
ttttttgtgg ttataaaatt tatatatttt attgtagaaa 18360atttagaaaa taagaggaaa
agtttaaaga agaaaattgt tataattttt ttatttaaag 18420atgatttgtt atttggtata
aaatttttag tgtgttttga gtattttttt tcgtattatg 18480tattttatag tttgttttat
tttatgtcgt gatatgtggt gattaattgt ttatgtgatt 18540ttatttttta ttttgttggt
ttattaggtg tttttttgag ttattgtttt ttattggttt 18600tgtttttggg tattttgttt
ttttttattt ttttattgta gtgagtatcg ttgtggagga 18660gtttgttttt gtttttgttt
ttgtgttatt tttaggatta tattttggag atgaaaaggt 18720gaaattaaat gttgtaaacg
gttttaagaa tgtggatata tagtagtaaa ttgttttttt 18780taacgattgt ttaaagtaag
ttatttttgt gtatattatt ttttatagat atttttattt 18840ttttttgggt attataaatt
aaatgattat ttttattatt ttattatgtt tttattttat 18900gttatatatt tatatttttt
ttgttattag taagttgaat ttttttatgt gtttatttat 18960tatttttgtg tgtgtgtgtt
tgtgtgtgaa atgttatttt attttttttg tttttttttt 19020gtttaagatg tttttttttt
tttattggta tgtaatgata ttgttttgaa aatttgatat 19080ttttttaatt ttagttatgt
ttttatgacg tttatatttt taaatttatt ttttttttat 19140gtttagaata agtatttatg
tatatatgtt gtgattattt tatgatttta ttgtaaaaat 19200ttaaattttg aattttttta
gaatttattt tattaatggg gatttaaata ggaaattaat 19260tttttttttt ttttaaatag
tttattattt gttttaatag tatttattga ataatcgttt 19320tttatagatt ttaaagaata
tgtttatttt ataataaatt tttatatata tttatatttt 19380ttgggttttg tttttttttt
ttttatttta tttaattatt tttggaattt tttgggtttt 19440ttattttgat ttttgtgttt
gttttttttt ttttaaagat agggttttat tttgttgttt 19500aggttggagt gtagtggtgt
tattatggtt tattgtagtt ttaatttttt tgggtttaag 19560tgatttttta ttttagtttt
ttaagtagta gagattatag ggatatatta ttatatttat 19620ttaatttttg tattttttgt
agagatgggt ttttattatg ttgtttaggt tggttttgaa 19680tttttgagtt taagcgatgt
gtttgtttta gttttttaaa ttgttgagat tatagaattg 19740agatattgtg ttttttttat
ttttttattt atatatgagt aatatattat tttaagtagc 19800gttattttat aattaatttt
agtaatttta tagaataaat gtttttattt ttattgttaa 19860aagataaaat tataataaat
ttagtttaaa gattttaatt gatttttttt tttttttttt 19920ttttgagacg gagttttatt
tttttgttta ggttagagtg cggtggtata attttggttt 19980attgtaattt tcgtttttta
ggtttaagtg atttttttat tttagttttt cgagtatttg 20040ggattattgg cgtgtgtcgt
tacgtttagt taattttttg gtattttagt agagaatggg 20100ttttattatg ttgtttaggg
tggttttaat tgtttgagtt taggtaattt atttattttc 20160gttttttaaa gtgttaggat
tataggtatg agttattacg tttggttttt aattaatttt 20220tatttgtgag tttataatta
gggaatattt tattttataa aatagatcga gtgttttgta 20280gagttgatta gagaagtttg
gttttttttt tttttgagac ggagtttcgt tttgttattt 20340aggttggagt gtagtggtta
gatttcggtt tattgtaagg ttcgtttttt gggttcgtgt 20400tatttttttg ttttagattt
tcgagtagtt gggattatag gtatttatta ttatattcgg 20460ttaatttttt gtatttttag
tagagacggg gttttatcgt gttagttagg atggtttcga 20520ttttttgatt ttatgattta
ttcgttttgg ttttttagag tgttgggatt ataggcgtga 20580gttatcgtgt ttggtagaga
agtttggttt tatagataga aaagagttag aaaaagtaga 20640aatagagaat aaaaagtaaa
ttggtttttt cgaagtgatt ttgtttataa gttaaagtag 20700aggggatttt tttattatgt
tagttaaaat tggtttgttg ggtatttggt tattattttt 20760tatttttttt taatttttta
gaaggttggt taagtaattt agttttggtt tagtggtaga 20820gaattttagt atgggcgatt
gtattttggt ttggtttgtt gggtttagtg tagtttagtt 20880taaataaatg gtttttttaa
tatttttttt aatattattt ttttttttaa aagtttttag 20940aaattattat gttaaggttt
gtttgtttat ttagtttatt ttaaaaagat gataaagtta 21000attagatatt tttttttgtt
ttataatttt agtttagatt aattggtttg tttagttatg 21060agttttatat tataattttt
tagtagagtt tagtcgagat tttggtatat agtatttagt 21120taaggattat tttttgaatt
aatttatttt ttattttgat ttttttttat attatagaat 21180gtttttttgg gtagcgttgt
gatttttgtt attttcgtga ttttatgtaa tgtattatgt 21240tattttatat ttaatgaggg
agttttagga gatttaatta ggagtaagtt ttggtttttt 21300aatgtttatt atgttattgt
agtttggtag atggatttgt ttgtagatga aagtttttgt 21360gtttttgttt gtttgttttt
ttgttttttg agatagttat gttttgtttt ttaggttgga 21420gtgtagtggt attattttag
tttattgtaa attttttttt tcgggtttta gtgattttat 21480ttttttagtt ttttaagtag
ttgggattat aggtgtgtgt tattatgttt ggttaatttt 21540tgtatttttg gtaaagatgg
ggttttgtta cgttggttag gttggtttcg aatttttgat 21600tttaggttat ttgttcgtta
tagtttttta aagtgttggg attataggtg tgagttattg 21660tgtttggttt tatttgggta
gttttttaag aatatagtag tttgggtttt gtatttagag 21720atattgattt tattgtttta
gggtaaggtt gagatatggg tggtttttaa gtatttaaga 21780tgttttatat gtgtatttag
ggttgaaaat tatcgatgta aagtaaaggg aagtgtagaa 21840gtgagatttt tatttagaga
taatttagta ggaaaaaatg tggttttaag tatttatagg 21900tttatatggt tatttaattt
aataaaaggg ttttattttt ttttgaaata tttgttttga 21960aataaaattt agaaggatat
aagttttttg taagaaaata ttattagtag tatgaattga 22020atagtattgg aattagtcgt
gggtgaatat aggagtatta tttttagata tttttaaaat 22080aattgagttt tttttttttt
gggaggggtg tagatagtag gggtaggatt taatgttttg 22140ataggttttg tttttattgt
atgatttgta ttgatgatgt tgtgtgtatt attttagaat 22200gtatggattt taaaggaaat
aaatatttaa taaattcgga gtggtagatt gataattgtg 22260agatatgtat ttgttacgaa
atagaaattt tatgttgtat tttgtaagtt ttaagttagt 22320gttggttaag agggggatat
agttgttttt tgaggaatag ttatttcgtt ttgtaaaatt 22380ttaattttta ggggtttagt
ttttgtttta gttttttata gattttatat agagtagttt 22440tagatttata gtaaaattga
gtagaggtat agaaattttt tatgtgtttt ttgtttttat 22500atatgtacga ttttttcgtt
attaatatcg tttattatag tggtgtattt gttatgattg 22560atgaatttat tttgtatatt
attattattt gagtttatag tttatgttag ggtttttttt 22620tggtgttgtg gtattggggg
gttagttttt agttttattt tttttttttt aaaaaattgt 22680gggttagtta taatggttta
cgtttgtaat tttagtattt tggggggtcg aagcgggcgg 22740attacgaggt taggagatcg
agattttttt agttaatacg tgaaatttcg tttttattaa 22800aaatattaaa aaaattagtc
gggtttggtg gcgggtgttt gtagttttag ttatttggga 22860ggttgaggta ggagaatggc
gtgaattcgg gaggtagagt ttgtagtgag tcgagatcgt 22920attattgtat tttagtttgg
gcgatagagc gagattgtgt ttaaaaaaaa aaaaaattgc 22980ggtaaaatat acgtaacgta
aaatgtatta tgttaataat tttaaatgta tattttagtg 23040gtattaggtt tatttatatt
gttgtggtat tattattatt tatttttaga atttgtttat 23100tttgtaaaat tgaagtttta
tatttattaa gtagttattt tttatttttt tttttttagt 23160ttttggtaat tattatttta
tttttgtttt tatgattttg attattttag gttttttata 23220taagtggaat tatatagtaa
ttgttttttt gagattggtt tattttattt agtataatat 23280ttttatggtt tatttgtgtt
gacgtttgta ttaatattgt ttttttgttc gaggttgaat 23340tatgttttgt aatatggaga
gattatattt tgtattttta tttatttgtt gatgaatatt 23400tgggttgttt tttattttaa
ttttattttt tttttgttgt ttttgaaatt tgataagtag 23460ttttatttta gatgggtttt
tgaagatagg aaagagtatt taattgtttg atattatttt 23520agagattggt tcggttatta
ttttagtttg ggttttgtag atttgtagaa atagtagtgg 23580ttttgagtat aggtagaggg
attagatgtg aggggtaagg gaggatttgt agtgtgtttt 23640gtattataaa atttatgttg
ttttattgtt taagtttaat ttacgttagt ttaaaggtta 23700tgtagatgtt ttttgttggg
ttgttatgga tattagaagt ttagaatttg agtaggcgga 23760atatagagta ttcgtagtgt
cgttttaatt tatttgtttt tttagtagtt atgtttataa 23820tagttattta atttttaatt
ttttaaattt atttaatttt tttatgtagt tttggtttgt 23880agtgttttgt aggaatagga
atggatttga tggtagagta ttggtgttaa ggggtatttc 23940gtaaatttgt ttgattatag
atttatttgg gaagtttgat gaaatataga tttttgggtt 24000ttattttaga attgattgat
tagaatttgt aggggagggg ttaaggaatt tgaattttaa 24060taggtgtttc gagagtttta
tgaggatatt gcgagtaaag aagtaagtat ttgatttaga 24120gtagaatatt tgagttttat
tgaatttgaa gtgtttggag agttttagtt atatttatcg 24180agtaggtttt atgttaggtg
tttaatggaa tagaggaata ttggattggg agatggttgg 24240ttgtttagag taggtttttg
ggtatcgtat tggttagatt attgggtatt atagttgaat 24300tttgattttg tggttttaga
aatttgatat tgtttttata ttaagtgttg atgttatttt 24360ggtggagtgt gattgtattt
tagttaggtg aaggattggt gattggtttt ttttttatat 24420aggagcgtgg ttttttggtg
gttaaaatgt ttatgttatt tagatttggt atttttgttt 24480ttttttaaga ttttttattg
taggttggga ttttcgtggt tttggtgtgg aaacgagtta 24540ggttgttttt tgtagtgaag
agggaagaat tttattgttg gagttagagt aatttgagtt 24600tgaattttta tttttttgta
tattagttgt gtgattttga gttagttttt gaatgttttt 24660gagtttttgt ttttttatat
tataattatg tggttatgat aattaaatga gattttgtgt 24720atatattttt taatttaata
aatatttaat gttttttttg tgttaatttt gtgttttgtg 24780attttgggat agtaattgat
atttggttag gttttaaggt tttaaggttt ttattgttat 24840gttgtttgtt ttatttggta
tattgtttta tgtaggggaa atgaattaat attttttttt 24900ttggaattgt tttatttttt
ttgttttttt tatgaatttt tttttttttt atggaaaaat 24960atagattttg attaagtttt
ttttggaaga agtttttgga tttattgtat tttttatggg 25020acgttgttta gattttaagg
tgttcggtgg tagtggagtt tgaggttggg ggtttaggta 25080gaggttttta ggtagggtat
gggagatgtt ttgggtttta cgttgttcgg gtttagggtt 25140ggttttttga tttgggtgag
gaaaagggag tgggagataa aaggttaagg ttatttagtt 25200attgattttg gttagttgaa
tattcgatgt tgatagtttt tttttttttt tttttttttg 25260ttgttttttt tatagagtta
agtttggttt gtattgaaat aagtatgtag taatatgttt 25320tttgttttat taatttgtat
ttgtatatag gtagtataga tttattattt tattttttat 25380tatgaattaa aagaattgta
tttttttaat aaatattatt ttatttttaa tttttttttt 25440ttattgtggt aaaatgtata
ttatataaaa tgtgttagtg taattatttt aaaatatata 25500atttagcggt attaagtata
tttatagtat tatgtaatta taaatattat ttttaaattt 25560ttttaatgat tttaatttga
aattttatat ttattaaata ataatttaga ttttttgttt 25620tttaaatttg tggtaatttt
aagtttattt tttgttttta tgaatttgtt tattttagat 25680attttatata aatataatta
tattgtgttt gttttttcgt gtttggttta ttttatttag 25740tatgttgtta aggtttattt
atgttgtagt atgtattagt attttatttt tgtttttgtt 25800agaataatat tttatggtat
ggttatatta tattttgttt atttatttat ttgttgaata 25860tttgagttgt ttttgtttgt
tggataaagt aagtaatgtt gttttgaata ttggtatata 25920gtgtttgtta atatataaat
attggtatat aatgtttgag tttttgtttt taattttttt 25980gggatatata tttaggagta
gttttttggg ttatatggta attttttgtt taatttattg 26040aggagttgtt aagttgtttt
ttatagtagt tgtattattt tatattttta ttagttagta 26100attattttta aatatgaaaa
atatgaaagg ttttaaatga tattttttaa ttttttaagt 26160aaatatatga attatagaat
tattgggaag tataagagaa aaaagaagaa atatttattt 26220ataattttaa ttgtgtaaag
gtaataatta ttaatatttt atttttattt atttatttat 26280ttgtattaaa aaaattaaga
tagggtttta ttatgttgtt taagttggtt ttaaattttt 26340gggattaagt tgttgtttta
ttttagtttt aagtagttgg gattataggt atgtgttatt 26400atgtttgggt ttttattatt
atttattttt tttttttttt ttttgagatg gaattttgtt 26460ttgtcgttta ggatggagtg
tagtggtgtg atttttgttt attgtaagtt tcgttttttg 26520ggtttacgtt atttttttgt
tttagttttt tgagtagttg ggattatagg cgtttgtaat 26580tacgttcggt taattttttg
tatttttagt ggagacgggg ttttatcggt ttagttagga 26640tggtttcgat tttttgattt
cgtgattcgt tcgtttcggt tttttaaggt gttgggatta 26700taggcgtgag atatagcgtt
cggttttatt attatttttt tatgtttttt ttttatatgt 26760tttttttttt atatagtgga
tattaaattt tatattatag ttttgtgggt tgattttttt 26820atttaatatt ttagtattgg
gttgggtatg gtgttttacg tttgtaattt tagtattttg 26880ggaggataag gtgggtggat
tatttgaggt taggagttcg agatttgttt ggttaatatg 26940gtgagatttt tttcgcgtcg
attcgttatt tttattaaaa atataaaaat tagttgggtg 27000tagtggtgtg tttttgtaat
tttagttatt taagaggttg aggtaggaga atcgtttaaa 27060tttgggagac ggaggttgaa
gtgagttgag atcgtgttat tgtattttat tttgggtgat 27120agagtgaaat ttattttaaa
aaattaaagt aataatagta ataatatata aatattttag 27180tattagtatt attttatttt
attagaattt tttaaaatat ttgtatgtat tgtagtttaa 27240ttagaggtgg tttttgattt
ttattagttt ttttttgggt tttatgatat tggtttggag 27300tgatttattg ttttggttgg
ttgagagagt ttttttgtta ataggttttt aagttagggt 27360tgggatttgt ttttttttta
tattttagta ataatatgtt ggttattttt tgaataggga 27420gggtgggagg agttagtatg
gaataagttg ttatttttta gagtagttag atttgttttt 27480gggtttgtta gtatttattg
atgatttttt gtgtttgtag gatggaatgt aggggtgagt 27540tgtgtgattt tatggtatag
attggtttag gtttttggag gttatatttt ggggttgtta 27600gttaaagggt gtttttaagt
tggagtgtgg gtgatttttt gaatagagtt ggtattgggg 27660agggggtgtt cggggttttt
ttttttttaa taggttttag ttttgtttta atgatagtgt 27720ttttatattt gtgggttatg
ataaagataa ttgttaaaga atttttaaga aggaggattg 27780taagtatatc gtggtggaga
agaaggattt aaaaaagatt tgttttgtta gtgaatggat 27840aatttaatgt gtttttagta
ggtatagggt ttttaggtta ggttttattt ttttttggtt 27900tttaatagtt aatgattgtg
tagttatgtt tattagtaaa aagatttttg agtaaatatt 27960tgaatatgtg tgttttttta
atttataatt tatgtatgta ttgatttaat atatattata 28020aagaatataa aatatttaaa
aagggagata aaattatata tgaatggtcg ggtatagtgg 28080tttatgttta taattttagt
attttgggag gttaaggtag gcggattatt tgaggttagg 28140ggatcgagaa tagtttggtt
aatatagtga aatttcgttt ttattgaaat ataaaattag 28200cgtggtggta ggtatttgta
gttttagtta ttcgggaggt tgaggtagga gaattatttg 28260aattcgggag gcggaggttg
tagtgagtta agattatatt agtgtatttt agtttgggtg 28320atagagtgag atttcgtttt
aaaaaataag taaataaata aataaattta aatttaaata 28380aaataaaata aaataaaata
tatatgaata aaagttttat tttttttaaa ttttataata 28440ggttattgtg tattttttat
ttaggggatt attgttattt ttattgtttt tgatgtttgt 28500attttgttgt tcgtgttttg
ttgtttggtg aacgatgaaa aaaatgtgtg tttttaagtt 28560ggaattagat tttttttttt
ttttacggtg tttttgttta tttttttttt ttttttatta 28620ggaagtgttt tttatgatgt
gttttttata tttttttgtg tttgtttgag gttaaagtag 28680tttagaataa gaaatagttt
agaataagtt tgtaaaatag tttagaataa gtttgaagaa 28740agtgttttta agattatatg
tagttcgagg ggaattaaag gttaggggtt ttgttttgta 28800gatggagagt ggtttgggtg
taaggtttta gtattagtta ttttttgtta tatatagatt 28860atcgaaaatt tagtggttta
agataacgat aatattgatt tttttttaat tgtttttgtg 28920ttaaagaata tttgtttatt
taataggttt gtattgagta tttgggttag gtagtgtttt 28980agatttgggg atatagtagt
gaattagaga gattttagga gagaattttg tttttttggg 29040gtttggattt agttgtgtgt
acgtgataga ttagaaatga gtaaatagtt atttgttgaa 29100taattagtgg gtaattgata
aagagtttta tttttttttg ttttttattt gtttaaatat 29160gttatgtggg ttagttgagt
aatttagata ggttttttag tttttttgtt tatttttaga 29220taatttaaaa aagtggtttg
agttatattt tgagttatat aaagttttat tttaggggtt 29280gtatttgaat tgagagggaa
tgggggatag gagagttaag ggtttttttt gtttgaggtt 29340atgttttagg ttgatttttt
tatgggattt ttgtgggttt ttttttttat agtttttttt 29400ttatttatag tttatgattg
ttattttagt ttttgtatcg taatttatgt tttttattgt 29460tttttaatga ggttgttttg
ttttttgtag tagagttatt tttggatgag atttataaaa 29520ttttgttatg attatttttt
ttgttgcgga atgtggggaa gtagttggta attgagtatg 29580gaggattttt gttaaaattg
agatagaaag aagtgttatt taatatttag ttataagtaa 29640ataatgtggg ttataatttg
ggttgtaggt tggacgtggt ggtttatgtt tgtaatttta 29700gattaggtgg gaggatcgtt
tgtgtttagg attttgagat tagtttgggt aatatagtaa 29760gattttgttt ttataaaaaa
atttaaaaat cgggtattat ggcgtatgtt tgtaatttta 29820gttatttagt aggttgaggt
aggagaatcg tttgaatata ggaggtagag gttatagtaa 29880gtcgagattg cgttattgcg
tttgagcgat agagttagat tttgttttaa agaaaaaaga 29940aaagaaaaaa agaatttggg
ttttgttatt tatttattta tttaattttt tttttttttt 30000729999DNAHomo sapiens
7aaaaaaaaaa aaaaattgag taaataaata aatggcagaa cccagattct ttttttcttt
60tcttttttct ttgagacagg gtctgactct gtcgctcagg cgcagtggcg caatctcggc
120ttgctgtaac ctctgcctcc tgtgttcaag cgattctcct gcctcaacct gctgagtagc
180tgggattaca gacatgcgcc atgatgcccg gtttttaaat ttttttgtag agacagggtc
240ttgctgtgtt acccagactg gtctcaaagt cctgggcaca agcgatcctc ccacctgatc
300taggattaca agcatgagcc accacgtcca gcctacagcc cagattataa cccacattat
360ttacttgtga ctagatgtta aatagcactt ctttctgtct cagttttgac agaggtcctc
420catactcagt tgccaactgc ttccccacat tccgcagcaa aggaggtgat catagcaggg
480ttttatggat ctcatccaag agtgactctg ctacaagggg caaggcaacc tcattagaaa
540acaataggag acatagatta cgatgcagaa gctaagataa cagtcatagg ctgtgagtgg
600aaggggagct gtggagagag aagcccacaa aagtcccatg aggaagtcag cctagaacat
660gacctcaggc agaaggagcc cttaactctc ctgtccccca ttccctctca attcagatgc
720agcccctgga atgagacttt gtgtaactca aaatgtaact cagaccactt ttttaaatta
780tctgaaggtg aacaagaaag ctaagggacc tgtctagatt gctcaactgg cccacatagc
840atgtttaaac agatagggga caggggaaaa taaagctctt tatcagttgc ccactgattg
900ttcaacagat gactgtttgc tcatttctga tctgtcacgt acacacagct gagtccaagc
960cccaggagaa cagggttctc tcctgaaatc tctctggttc actgctgtat ccccaggtct
1020agagcactgc ctggcccagg tgctcagtac agacctgttg agtgaacaga tgttctttaa
1080cacagaaaca gttaaaagag aatcaatgtt gtcgttgtct taagccacta agttttcggt
1140ggtctgtgtg tagcaagagg tagctgatgc tggggccttg cacccaggcc actctccatc
1200tgcaaggcag agcccctggc ctttaattcc cctcgggctg catgtggtct tgggggcact
1260ttcttcaggc ttgttctaag ctgttttaca ggcttgttct aagctgtttc ttgttctaag
1320ctgctttgac ctcaagcaaa cacagaggaa tgtgggggac acatcataga agacacttcc
1380tgatgaagag gaaggaggga taaacaggaa caccgtaaag aaaagaaggg atctaattcc
1440agcttgaaga cacacatttt tttcatcgtt caccagacag caggacacgg gcagcagagt
1500gcagacatca gaaacagtaa gagtggcagt ggtcccctga gtaagggatg cacaatgacc
1560tattatagag tttaggaaaa atagaacttt tattcatatg tattttgttt tgttttgttt
1620tgtttgggtt tgggtttgtt tgtttgtttg cttgtttttt gagacggagt ctcactctgt
1680cacccaggct ggagtgcact gatgtgatct tggctcactg caacctccgc ctcccgggtt
1740caagtgattc tcctgcctca gcctcccgag tagctgggac tacaggtgcc tgccaccacg
1800ctaattttgt atttcagtgg agacggggtt tcactatgtt ggccaggctg ttctcgatcc
1860cctgacctca aatgatccgc ctgccttggc ctcccaaagt gctgggatta taggcatgag
1920ccactgtgcc cggccattca tatgtagttt tgtctccctt tttaaatgtt ttgtattctt
1980tataatgtgt attaaatcaa tgcatacata aattataaat taaaaggaca cacatattca
2040agtgtttgct caaaaatctt tttactgata ggcatggcta cacaatcatt gactattaga
2100ggccagagga gaatgaggcc tggcctggga gccctgtgcc tactagaagc acattagatt
2160atccattcac tgacagaaca ggtctttttt gggtccttct tctccaccac gatatacttg
2220cagtcctcct tcttgaagat tctttggcag ttgtctttgt cataacccac aggtgtagaa
2280acactgtcat tgagacaaaa ctggggcctg ttagaagaga aaggaccccg agcaccccct
2340ccccagtgcc agctctgttc aggaagtcac ccacactcca acttaagggc accctttggc
2400tggcagcccc agagtgtgac ctccaggaac ctggaccagt ctgtaccata gagtcacaca
2460gctcacccct acattccatc ctgcaggcac agaaggtcat cagtaggtgc tgacaggccc
2520aggggcaagt ctggctactc tagaaagtgg cagcttgttc catgctggct cctcccaccc
2580tccctgttca ggaagtggcc agcatattgt tgctagaatg tggaaaggaa acaaatccca
2640gccctgactt ggaggcctgt tggcaaagga gctctctcaa ccaaccagag caatgagtca
2700ctccaggcca gtgtcatgga gcccagggag aagctgatga aagtcagaga ccacctctaa
2760ttgagctgca gtacatacaa atattttgaa gagttctgat gagatgagat gatactaatg
2820ctagaatatt tatgtattat tactattatt actttaattt tttgagatga atttcactct
2880gtcacccagg atggagtgca gtggcacgat ctcagctcac ttcagcctcc gtctcccagg
2940tttaagcgat tctcctgcct cagcctcttg agtagctggg attacaggag cacaccacta
3000cacccagcta atttttatat ttttagtaga gatggcgggt cggcgcgggg gaggtctcac
3060catgttggcc aggcaggtct cgaactcctg acctcaaatg atccacccac cttgtcctcc
3120caaagtgctg ggattacagg cgtgagacac catgcccagc ccagtgctag aatattaagt
3180ggaaaaatca gcccacaaaa ctgtgatata aaatttgata tccactatgt aaaagaaaaa
3240acatatagaa aagagacata aagaaatgat gatagggccg ggcgctgtgt ctcacgcctg
3300taatcccagc accttgggag gccgaggcgg gcggatcacg aggtcaggag atcgagacca
3360tcctggctaa accggtgaaa ccccgtctcc actaaaaata caaaaaatta gccgggcgtg
3420gttgcaggcg cctgtagtcc cagctactca ggaggctgag acaggagaat ggcgtgaacc
3480caggaggcgg agcttgcagt gagcagagat cacaccactg cactccatcc tgggcgacag
3540agcaagattc catctcaaaa aaaaaaaaaa gaaaataaat gatgatagaa gcccaggcat
3600ggtggcacat gcctgtaatc ccagctactt ggggctgagg tgagacaaca gcttgatccc
3660aggagtttga gaccagcttg ggcaacatag tgagaccctg tcttaatttt tttaatacaa
3720ataaataaat aaataaaaat agaatgttaa tagttattac ctttacacag ttgggattat
3780gagtaggtat ttcttctttt ttctcttatg cttcccaatg attctataat tcatatgttt
3840acttagaaaa ttgggaaata tcatttaaaa cctttcatgt ttttcatgtt taaaggtgat
3900tgctggctgg taggaatgta aaatggtgca gctgctgtgg aaaacagctt ggcagctcct
3960caataagtta aacaaaaaat taccatatga cccagaaagc tactcctggg tatatatccc
4020aaaagaattg aaagcaggga ctcaaacatt atatgccaat gtttgtatat tgacaaacac
4080tgtatgccaa tgttcagaac aacattactt actttatcca acaggcagaa gcaactcaag
4140tgttcaacaa ataaatggat aagcaaaatg tggtatgacc atgccatgga atattattct
4200ggcagaaaca agaatgaagt actgatacat gctacaacat gaatgaacct tgacaacatg
4260ctgagtgaaa taagccagac acgaaaagac aaacacagtg tgattgtatt tatgtgaaat
4320atctagagta ggcaaattca tagagacaga aagtagactt gagattacca caggtttggg
4380ggacagggaa tctgagttat tgtttaatgg atatagagtt tcagattgga gtcattaaaa
4440agatttggaa atagtgttta tggttgcata gtactgtgaa tgtacttaat gccgctgaat
4500tgtatgtttt aaaatggtta cactggcaca ttttatgtag tatacatttt accacaataa
4560aaaaaaaagg ttaaaaataa agtggtgttt gttgaagaga tacaattctt ttagttcatg
4620gtaggaagtg aaatggtgaa tctgtgctgc ctgtgtgcaa atacaagttg gtaaggcaaa
4680aggcatatta ctgcatgctt atttcaatgc aaaccagact tagctctata gaggaaacag
4740cagagagaga gagggaggga gaaactgtca acatcgggta ttcagctaac cagagtcagt
4800ggctggatga ccttggcctt ttgtctccca ctcccttttc ctcacccaag tcagggagcc
4860agccctgggc ccgggcagcg tggagcccag gacatctccc atgccctacc tgggagcctc
4920tgcctgggcc cccagcctca ggctccactg ccaccgagca ccttgagatc tgggcagcgt
4980cccatagagg gtgcagtggg tccaaaaact tcttccagaa aaagcttaat caaagtctgt
5040atttttccat ggaggagaaa aaagttcatg agaaaaacaa aggaggtgag gcaattccag
5100ggaagaaggt gttgattcat ttcccctgca tggagcagtg tgccaaatgg aacaggcaac
5160atggcagtag aaaccttgaa accttgaaac ctgaccaggt gtcagttact gtcccaggat
5220cacaggacac agaattggca cagagaaggc attaagtgtt tgttgggtta aagaatatat
5280acacagaatc tcatttaatt atcatagcca cataattata atgtgaggaa acagaggctc
5340agagacattc agaaactgac tcagggtcac acagctagta tacaagggaa tggagattca
5400gactcaagtt actctgactc caacagtgag gttcttccct cttcactgca aggagcagcc
5460tggctcgttt ccacaccagg accacgagaa tcccagccta cagtggagaa tcttagaaga
5520gggcagggat gccaaatctg ggtgacatgg acattttagc caccagggag ccacgctcct
5580atgtgaggga gggaccagtc accagtcctt cacctggctg ggatacaatc acactccacc
5640agagtgacat caacacttga tataaagaca atgtcaaatt tctggagcca caaaatcaag
5700gttcagctgt aatacccagt gatctggcca atgcgatgcc caggagcctg ctctgagcag
5760ccagccatct cccagtccaa tattcctctg ttccattagg cacctagcat gaggcctgct
5820cgataaatgt ggctggaact ctccaggcac ttcaagttca gtaggactca ggtgttctgc
5880tctaagtcaa gtgcttgctt ctttactcgc agtgtcctca taagactctc gaggcacctg
5940ttaaaattca gattccttgg cccctcccct acagattctg atcaatcagt tctgggatgg
6000agcccaggaa tctgtatttc atcaagcttc ccaggtgagt ctgtgatcag acaagtttgc
6060gaaatgcccc ttgacaccaa tgctctgcca tcagatccat tcctgttcct gcagaacact
6120acaagccaag gctacatgaa gaggttagat ggatttgggg ggttgggagt taggtagctg
6180ttatggacat agctgctgag gaaacaggta aattaggacg gcactgcggg tactctgtgt
6240tccgcctgct caagttctag acttctggtg tccatagcaa cccagcagaa aacatctgca
6300tagcctttgg gctgacgtga gttaaacttg agcagtgaag cagcataagt tttatggtgc
6360aaggcacact acagatcctc ccttacccct cacatctgat ccctctgcct atgctcagag
6420ccactgctat ttctacaaat ctacaaggcc cagactgagg tgatgaccgg gccaatctct
6480ggaatagtgt cagacaatta gatactcttt cctgtcttca ggagcccatc tggaataaag
6540ctacttatca agtttcaaaa acaacagaaa agaaatgaga ttgaggtgga aaacaaccca
6600agtgttcatc aacagatgaa tggagatgca aaatgtggtc tctccatatt acagaacatg
6660attcagcctc gaacaagaag gcaatgttga tgcaagcgtc aacacagatg aaccatgagg
6720atattatgct aagtgaaatg agccagtctc aaaaaagcaa ttactgtatg attccactta
6780tatgagggac ctagagtagt caaaatcata gagacagaag tgaaatggtg gttgccaggg
6840gctggggaga aggggatgga gagtgactgc ttaatgggta tagagcttca gttttgcagg
6900atgaacaagt tctggaggtg gatggtagtg atgccacaac aatgtgaatg gacctaatgc
6960cactaaagta tacatttaaa attgttaaca tggtacattt tacgttacgt atattttacc
7020gcaatttttt ttttttttag acacagtctc gctctgtcgc ccaggctgga gtgcagtggt
7080gcgatctcgg ctcactgcaa gctctgcctc ccgggttcac gccattctcc tgcctcagcc
7140tcccaagtag ctgggactac aggcacccgc caccaagccc ggctaatttt tttggtattt
7200ttagtagaga cggggtttca cgtgttagct aggagggtct cgatctcctg acctcgtgat
7260ccgcccgctt cggcccccca aaatgctggg attacaggcg tgagccattg tgactggccc
7320acaatttttt aaaaagaaag aaataggact gaaaactaac cccccaatac cacaacacca
7380agaggaagcc ctaacataaa ctatggactc aggtgataat gatgtgcaag gtaggttcat
7440cagtcataac aaatacacca ctgtggtgga cgatgttgat aacggggagg tcgtgcatgt
7500gtgagggcag gaggcacatg ggaaatttct gtacctctgc tcagttttgc tgtgaatcta
7560aaactactct atataaagtc tataaaaaac taaaacaaaa actaaacccc tgaagattgg
7620aattttgcaa gacggagtag ctgttcctca gagaacagct atgtccccct cttggccagc
7680actggcttga gacttacagg gtgcaacatg aaatttctgt ttcgtagcaa gtgcatgtct
7740cacagttgtc agtctgccac tccgagttta ttgggtgttt gtttcctttg agatccatgc
7800attctaaaat aatacacaca acatcatcag tgcaagtcat gcaatgagaa caaggcctat
7860caggacattg agtcctgccc ctactatcta cacccctccc agagagagag gagctcaatt
7920attttaaagg tatctaagaa tggtgctcct gtattcaccc acggctaatt ccagtactgt
7980tcagttcatg ctactgataa tgttttctta cagaaagctt atatccttct aagttttatt
8040tcagaacaga tatttcaaga aaaaataagg cccttttatt gaattagata gccatgtgag
8100cctgtgggta cttgggacca cattttttcc tactggatta tctctggatg agaatctcac
8160ttctacactt ccctttgctt tgcatcggtg gttttcaacc ctgggtgcac atgtgaaaca
8220tcttgggtac ttgaaagcca cccatgtctc agccttaccc tagaacaatg aaatcagtat
8280ctctgggtac agggcccagg ctactgtgtt cttagaaggc tgcccaagtg aggccaggca
8340cagtggctca cacctgtaat cccaacactt tgggaggctg tggcgggcag atgacctgag
8400gtcaggagtt cgagaccagc ctggccaacg tggcaaagcc ccatctttac caaaaataca
8460aaaattagcc aggcatgatg gcacacacct gtagtcccag ctacttggga ggctgaggag
8520gtagaatcac tggaacccgg gaggaggagt ttgcagtgag ctgaaatggt gccactgcac
8580tccagcctgg gagacagagc atgactatct caaaaaacaa agaaacaaac aaacagaaac
8640acaaaggctt tcatctgcag acaggtccat ctaccaggct gcaataacat aataaacatt
8700gaaaagccaa gacttactcc tggttgaatc tcctggaact ccctcattag gtatgaaata
8760gcatgatgca ttgcataaag tcacgaaggt ggcaaagatc acaacgctgc ccaggagaac
8820attctgtaat gtgaagaaag gtcagggtgg agaatgaatt aattcaaagg ataatccttg
8880actgagtgct gtgtaccagg atctcggctg ggctctgctg agaggttatg atgtggagct
8940cataactgaa caaaccagtt aatctaggct gaaattgtag aacaaggaaa agtatctagt
9000tgactttgtc atctttttga gatgagctaa ataaacaaac aaaccttaac atggtaattt
9060ctgggaactt ttgaaaggga agatggtgtt aaaaaaaatg ttgaggaggc catttgtttg
9120gactgagctg cactgggccc aacagaccaa accaaaatgc agtcgcccat gctgaagttc
9180tctgccacta agccaaaact aagttgctta accaaccttc taagaaatta gaagagagtg
9240agaggtaata gccaaatgcc caacaggcca gttttagctg acatgatgag aaagtcccct
9300ctgctttaac ttataagcaa agtcacttcg aaaagaccaa tttgcttttt gttctctgtt
9360tctgcttttt ctggctcttt tctgtctata aagccaaact tctctgccag gcacggtggc
9420tcacgcctgt gatcccagca ctctgggagg ccaaggcggg tggatcatga ggtcaggaga
9480tcgagaccat cctggctaac acggtgaaac cccgtctcta ctaaaaatac aaaaaattag
9540ccgggtgtgg tggtgggtgc ctgtagtccc agctactcgg gagtctgagg caggagaatg
9600gcacgaaccc aggaggcgga ccttgcagtg agccgagatc tggccactgc actccagcct
9660gggtgacaga gcgagactcc gtctcaaaaa aaaaaaaagc caaacttctc tggtcagctc
9720tgcagaacac tcgatctatt ttatggaatg aagtgttccc tgattataga ctcacaagta
9780aaagttaatt gagggccagg cgtggtggct catgcctgta atcctagcac tttgggaggc
9840ggaggtgggt ggattgcctg aactcaggca attgaaacca ccctgggcaa catgatgaaa
9900cccattctct actaaaatac caaaaaatta gctgggcgtg gcggcacacg ccagtaatcc
9960caggtactcg ggaggctgag gtgggagaat cacttgagcc tgggaggcgg aggttgcagt
10020gagccaagat tgtgccaccg cactctagcc tgggcaagag agtgagactc cgtctcaaaa
10080aaaaaaaaaa aaaaaaaatc aattaagatc tttaaactaa atttgttgta attttgtctt
10140ttgacaatgg gaatgaaggc atttattctg taagattgct aaaattaatt atgaagtgac
10200gctacttgaa gtagtatgtt actcatatat gaatagaaag atagggaggg cacagtgtct
10260caattctgta atctcagcaa tttgggaagc tgaggcaggc acatcgcttg agctcaggag
10320ttcaagacca gcctaggcaa catggtgaaa acccatctct acaaaaagta caaaagttag
10380gtgggtatgg tggtgtgtcc ctgtagtctc tgctacttgg gaggctgagg tggagaatca
10440cttgagccca ggaaggttga ggctgcagtg agccatgata gcaccactgc actccagcct
10500gggcaacaga gtgaaaccct gtctttaaaa aaaaaaaggc aaacacagag atcaaaatag
10560aaaacccaga aaattccaaa ggtagttgga tagggtaaaa aagaaaaaaa caaaacccag
10620aaagtataaa tatatgtaga agtttattat aaaataaaca tgttctttga gatctgtgga
10680gagcgattat tcaataaatg ctattagaac aagtggtgaa ctatttgggg aagaaaaaaa
10740gttaatttcc tatttgagtc cccattagtg aaataaattc taaaaggatt caaggtttaa
10800gtttttacaa taagatcata aaataatcac agcatatata cataaatact tattctaagc
10860ataaaagaaa aatagatttg agaatataaa cgtcataaga acataactga aattagaaag
10920atatcaaatt ttcagaacaa tatcattaca taccaatggg agaagaaaaa catcttaaac
10980agaaaaaggg caagagaaat aaagtggcat ttcacacaca gacacacaca cacagaaatg
11040atgaataaac acatgaaaaa gttcaactta ctgataacaa aagaaatgta aatatgtagc
11100atgaaataga agcataatgg gatggtgaga ataatcattt aatttataat acccagaggg
11160aaatagaggt atctatggga ggtgatatgc acaagaatga cttactttga acaatcgtta
11220aggagaacaa tttgctacta tatatccaca ttcttaaaac cgtttacaac atttgatttc
11280accttttcat ctccaagatg tgatcctaaa aatggcacag agacagagac agagacagac
11340tcctccacaa cgatgctcac tgcagtggaa aaatggaaag aaacaaaatg cccaagaaca
11400gagccagtgg agaacagtga ctcagaaaaa cacctggtga gccagcaagg tagaaggtga
11460agtcacatga gcaattgatc accacatgtc acgacatgaa atgaagcagg ctatgaaata
11520catgatgcgg agagagatgc tcagaacaca ctgagggttt tgtaccaaat aacaagtcat
11580ctttggatgg ggagattatg acaattttct tctttggact tttcctctta ttttctaaat
11640tttctacaat aaaatatatg agttttatag ccacaaagaa agaaagattc tttaaaaatg
11700acacaacgcc ttgatacatt cagctcaagc caggacttgg agtggcactt gttctgtttc
11760attcttctga gtagccttca gctcgaaatc attgtggaaa cctaaacttt tccccccaaa
11820aagccagcct tgttgacagc atttttactc ccttcatgca gaaagagggc tcctggggta
11880ctggggatca ctggaatctt ggggatctgg tttcactgag tcacaaagca ggagagtcaa
11940cagggacaag cagggctgca tcccaggaca agtgacatgt gcaaaatccc caagtctcct
12000gcagctctag agcagaggca ggagattgaa ccactggggg agagcccaaa catggtgaca
12060gcagagagca aattatttaa ttttgagatt atccacttga cagtatctca gaaatagcct
12120aatgctgtct ctctctctct ctccctccct ccctccctcc ctttctgtct ctctctctct
12180ctctctctga aactcagttt tccttccctg taaaaagagg ggttctccca tttgggtgac
12240cattagagtt atccagggag ctcataggaa acacaatcta ggagccagtg cctcagtgtc
12300tttacctagt ctcccaggtg agaacccagg aacaatgacc aacccagtgc tgctttaatg
12360tgcatataaa ttcctgggag ttttttttgt tttgttttgt ttgtctgttt gtttgtttct
12420ttttgggacg gaatctcgtt ctatcgccta gagtagagtt cagtgatgca atcttggctc
12480actgcaacct ccgcctcccg ggttcaagtg attctcctgc ctcagcctcc tgagtagctg
12540ggattacagg agtgcaccac cacacctggc taatttttgt atttgtagta gagacgtagt
12600ttcatcatgt tggccaggct ggtctcaaat acctgacctc aaatgatccg cccgcctcgg
12660cctcccaaag tgctgggatt atgggcgtga gccaccgcgc ccaaccagac agcctgttaa
12720ttaacctgca gattctgctt cagtaggtct ttagacttac acggaagctc ccaggtgatg
12780cccacgctgc tggtctgagg tccacccctg agtagagagg ctctaaagct tttttcattc
12840ctgtcttcta ttctgccacc agccttcctt ccctttgtgt cttcctttcc tgcatttagt
12900aaccctgagc tcctgatgtg tgcaggagat tacaccacga cgccagaatg ggctaggatt
12960ttctagatgg tacagcgagc cttgaggtga agcaggctgg cagtatcctt agaagttgtg
13020tctcttggct gggcgcggtg gctcacgcct gtaatcccag cactttggga ggccgaggcg
13080ggtggatcat gaggtcagga gatcgagacc atcctggcta acatggtgaa accccgtctc
13140tactaaaaat acaaaaaatt agctgggcgt ggtggcgggc gcctgtagtc ccagctactc
13200aggaggctga ggcaggagaa tggcgtgaac ccgggaggtg gagcttgcag tgagccgaga
13260ttgcgccact gcactccagc ctgggcgaca gagagactcc gtctcagaaa aaaaaaaaaa
13320aaaaaaaaaa aaaaagttgt atctctttag gtaaattact gctcctggaa gggttatccc
13380atgatcaaga gttgggggtc tggccaggca taatttctca cacctgtaat cccagcactt
13440tgggaagcca aggtgggagg atcacttggg cccaagagtt agagaccagc ctgggcaaca
13500tagtaaaaac tcatctctac tacaaataac aataataaaa taaataaata aataaataaa
13560gagttggggg tcctcaggat cttaggcagg cagaggtatt gccagctctg cagcttgcta
13620agttcactgt gaggctgcat agagtatagg ctggaggagc ctgtctagtg gagatgggga
13680ggacctccca ccccactgtt gttttaccat ccccagttct gcctccggac agtttctgag
13740agcaagtttg tcttgccgag tggcccagag gaggcccggg gtcccctcct gaggaccctc
13800tcccacctcc cttggctcct cccacttggc tggctgtttc ttgcctcatc tcccttgcag
13860cccccagagc agcccagttc agtccccagc gttagtaaag aatgttttaa gaaagatcaa
13920gtttaagaca caagaaaagc ctgcatgggt ttttataaat tttgaagcaa aattctttct
13980tggccaggca cagtggttca caccagtaat cccaccactt tgggaggctg aggtgggcag
14040attgcatgag cccaggagtt caagaccagc ctgggcagca tagcaaaatc ctatctctaa
14100aacaaataca aaaattagtc gggcatggtg gcactcgcct gtggtcccag ctacttggga
14160agctgagatg ggaggatcac ctgagcccag gaaggtccag gctgcagtga atcatgactg
14220caccactgca ctccagcctg ggcaacagag tgagatcctg tctcaaaaac aaacaaataa
14280accaacaaac acagccccaa acccaagatt tttccttaac acatgataca gtgaaaaggg
14340cctagggaaa tggttcccaa acttgagtat taaaatgacc tgggcagctt tttaaattga
14400tgctctgttc tcactgcagc ccaattaaat cagagtgtcg gggtggaaga tgggtgtcag
14460tgttcttcaa aattcccccg gagtttccac tgcacagcca agtttgggag cccctggcag
14520ggagagagga atgcctgggt tcctatctca gctcattcac aggcttgcgg tgagacccca
14580gtggcagctt cttcccctcc ccaggctcag ttccctcatc tcaaaaacga tgcagtcatg
14640tagctctgaa attattttta atttcaaatt actcacatag gacccaagaa ttagagacac
14700ttatgaaatc taattgaacc ccagaattac tggaaggaaa aacaacttag atcttagaga
14760acagaactag tgctaaactc attacaattt tcgcaagctc tcagactctc atactctgca
14820cattgaattg caggtaataa cataaggttc ctaactaggt cacatttacc attcaatgct
14880atgtggtaga agcacacgca tattaaaata ggaatacata ttctcttttg cttttctagc
14940ctttatatat aaaaccagtt acctaccatt gtgataagca ggactcctta tagacaggta
15000catccaggca aagctgcatc aaacttttat actggagagg gcaaccacgc attcgacatc
15060attgggaatg ataacaaggt gagaacacat tgagtgttta cagagggttg actatcaaaa
15120ggaaagtatg gtttcagatg atttatgagt tattaatcaa tcacatctcc acccaagtta
15180tgtgtttacc ttgtccccac cccgatctgc atttgctgct gacctaaaag agcagaggag
15240gagagaactt ctctttgctg agccttcgcc ctatgctggt ggattgctag cacgtgatgt
15300gtccttattc gattttatac acaaagaaac agcctcagcg atgccagtca caacagccag
15360gtggtaaagg tgggtttcct gaaggcataa gaatagccca caggtcagcc caccaggccc
15420accgggcctg ggtctgtggc ccagatgacc ctgcccttag ggtctgacat gagaacagaa
15480atgggtcaag ccatcctgca gggcacagga gagcaagaac tacatacagc tcggggttcc
15540ctgacaagct gaattgctgg acctttctag agcccttctt gaatctgtag tctactccct
15600gctgcatcaa gcaagccttg tttctctttg ttccacagtg gtctcctccc aaacaaccgc
15660acctgccctc atcatggcta ggatggcctt tggggaatgg caacaagcag aacctcctag
15720ggtgctggag ggacttacta ctttatctgt ttgctctgcc accacaacaa aacactgcaa
15780ctgggtggct ttgactaaag aaatttattt gctcacagtt ctggagacta gaaatgtgag
15840gtcaagccat tggggaaggc ttgttttcct ctgagccctc tctctttggt ttgcagatgg
15900ccatgctctt gctgcctctt acatggcatg catccctggc atctctctct gtgtccaaat
15960ttcctcttct gataaggaca ctagctgtat tggattaggg tccactctaa ctatctcatt
16020ttacctaagc cacctcttta aaggccgtgt ctccattaca gtcacattct gggatattgg
16080gggttagggc ttaaaccaat gaatttttag gggatgcaat tgagctcata agacaatttt
16140atcccaaaaa gtcccctcac tcctcgccac agcttgctac agttcctaaa atttaacctg
16200agatccctct gcccacagat gagagttcat gctcctaacc aggggatcct tgaagctcat
16260ctgagctggc cacagtggcc ctttccagca ccatcctcta ccctccctaa aatcagcctc
16320accaccccta acacagcttg aggtcactct cctgctcctt agaagagctg tagtgctgtc
16380tggctgcctc ctcactcacc aacggtgcca cacacttctg cctttacctg cataagttaa
16440gtgttcatcg actctaagtt ccaggaaatc ggggcacatt catcatgcac tgtagcccct
16500tggattcttt taaaactcca agtttgaaga acttcccaca gtgtgaagtg ggaagttctg
16560ccttacattg agaggaacta ggatttgctg ccctcacctg cttacctcca gggtacaggc
16620acaaacctcg gctccacaaa cactctcctc tcaaaaagtc ctcttcagag agggtccaag
16680gacactggca tcatgcagaa tctccctagg aatgatggtg tggagggttc tggctcttag
16740ggcaagggtt tcttggagtc cagtgtgcag tgtcagtgca tgggggctgt gactgtgact
16800ggtagcaagg tggtcttgct ggagccaagc agcagtgtgg tttcagtgct ctacctgaac
16860ttgtgccaag aaagcctgtg tctctggccg ttagtagagg cttgaattct ctaacaaatc
16920tctttccacc tcaaatgacc tagagtaaat tctattgttt gcagttaaga actttgtgac
16980ccagacagtg actgtgacct attcctgtct gtgtgccttc cagtgaccag cagggtgcct
17040ggtgcaaagc agctgcccag aacaggattt cacctgggtt atctcaggta ttcccaagta
17100gaaagtatcc tggcaagacc aacccatatc actgctgaaa tgagctcatc tactgctccc
17160caggggttga gaaagggatg ctcagagcag gtgtggccag ctgcccacag ggaggaaggc
17220aaatgaaatg agcagtgctc tccgggtgtg tgacagccag ctctgtccaa acctgcctga
17280tcagttttca gtgttgatcc tacctcaact gtcatcctta tagatggaac caacttaatt
17340tcttttaatt atttattttt ctttttattt attttaattt tttttaaaga catagtcttg
17400ctctgctgct caggctggag tgcagtggca ccattatagc tcactgttac ctccaattcc
17460tagattcaat tgatcctccc acctaagcct cccaagtagg tgggactaca ggagcgtgcc
17520accacaccca gctaattttt agattttttt tgtagagaca gggtcttgct atgctgccca
17580gcctggtctt gaactcctgg gctcaagtga tgctcctgcc tcctcctccc aaagtgttgg
17640gattacaggc ataagccacc atgcccaggc tgattatcat tttaatggct acattttatt
17700ccatggaatg atatggacca tcatttattt ggtcaggtac ctcttgttga acactcaggt
17760tgtttctgac ttttttttct ataattagaa acactgggct gggcatggtg gctcacacct
17820gtaatcccag cactttggga ggccgagacg ggcggatcac gaggtcagga gatcgagacc
17880atcctggcta acatggtgaa accccgtctc tactaaaaat acaaaaaaat tagccgggcg
17940tcatggcggg cgcctgtagt cccagctact cggaaggctg acgcaggaga atggcgtgaa
18000cccggaaggc ggagcttgca gtgagcggag atcgcgccac tgcactccag cctgggcgag
18060agagcgagac tctgtctcaa aaaaaaaaaa aagaaacgct gttgaaatcc aggtccttca
18120gataaaaatt tcccacactt atactaattt ctttaagata aattcataga ggccgggtga
18180ggtggctcgc acctgtaatc ccagcacttt ggaaggacga ggtgggtgga tcacgtgagg
18240tcacgagttc gagaccagcc tgaccaacat ggagaaaccc cgtctctact aaaaatacaa
18300aattagccgg gcgtggtgga gcatgcctgt aatcccagct actccggagg gtgaggcagg
18360agaatcactt gaaaccggga gacgaaggtt gctgtgagct gagatcacgc cattgcactc
18420caacctgggc aacaagagcg aaactccatc tcaaaaaaaa aaaaaatcac aaaggtagaa
18480tttctgggta aaagaaaatt ataattctct tagattatca cctgttacta tgatatgtgg
18540gaaatgcttt gtcttaattt atttttcttt gctaattggc aagattagac cttttttttt
18600tttttttttt ttttttttga gacggagtct cgctctgtct cccaggctgc agtgcaatgg
18660tgcaatttca gctcagtaca agctccacct tccaggttca cgcccttctc ctgcctcagc
18720ctcccgagta gctggaacta caggcaccct ccatcacacc cggctaattt tttgtatttt
18780taatagagac ggggtttcac catgttagtc aggatggtct tgatctcctg acctcgtgat
18840ctgcccccct cgggcttcca aagtgctggg attacaggcg tgagccaccg tgcacagcct
18900agaccttttt ttgtatatgt attgaccaat tgtttttctt cttttgtgag taccctgttc
18960actcatgtcc tttagctatt tttctgtttg ggttatttat atttttcttc tggattagtt
19020aaggcttcta atattaaaga tattaatctt caagtgtcac acatgttaaa atatttccct
19080gatttttttc atggtcagca cacccattct ggagttagaa ctaaggaatg agaaattttc
19140ctcatttata tttttacctc tcaaatgata tgttttgaca tgtagagatt ttaaacttta
19200actgactaca gccacactat ccccacctgt gggtcagcac tacaagcccc aaagaacacc
19260tcaagatcca tggagtcaca cagcccttgg gctgatgctg tggcaggagc tgccattcat
19320ttccagggtt ttttgttttg ttttgttttt tgtttttttc tgagacagag tcttgctctg
19380tcgcccagga tggagtgcag tgctgcaatc tcagctcact gcaacctcca cctcccgggc
19440acaagcgatt cttgtgcttc agccttccca gtagctgggg ctataggcat gcaccaccat
19500gcctggctaa tttttgtatt tttagtagag acagggtttc accattgccc aggttggtct
19560tgaacgcctg acctcaagtg atctgcctgc cttggtctcc caaagtgctg ggattacaag
19620catgggccac cgtgccctgc catcattccc agtgttaaaa gaacgttaga caaattaaat
19680ttaacagagt ttgattgagc aaagaaccat ttgtgtgaat caggcagccc ccaggcaaaa
19740ataagttcag agagactcct gcactgcctc atagtcaaag attatggaca gaaaaaggaa
19800agtgatgtac agaaactgga agtgaggtac agaaacagac ggattggtga tggcatggtg
19860cttggcttat ttgagtccag tttcaacagc tggccgcctg tgagtggttg aagtctggct
19920gctgtgatta actgagactc ggctacttgg tacgagagta ggtgacagcc tgtttacacg
19980tgcagttagc ttacagttca ctatgtacag agaaaccatt agggtgaatt taaaatatgt
20040aaggagaagg ctttaggcta aacttgattt aacaccagga tcggtggggt ctccccttac
20100cagtcttcaa gctgatgact tatctctcat ccacttcctc tcccatcgtg ctctagagcc
20160tttccaaacc atgatctctt ctcttaaacc tctcacccaa ctgcctactt catacgcaga
20220acacaatctt atttcgtatc tcacagagac cagaggtgaa aactgcaccc ccacccctac
20280cctgcacatt gccctccacc ggctcctgtt ctttccactt tccctctggg tcccttcctc
20340accccaaata cgcaatacgg atcccattcc cctccccatc ggcccccatt ccttcccagg
20400gacccctcca taaaagccag cctccccaag acaagcaggc atgaagggac agatggaaaa
20460gtgttcagtg ttcgaggact gcccaattcc cagcctggca taagccagcc tccctgaggg
20520tccacttata ccctggcaat tttactttgt agagcccaat ctttgtgata ccctgtggca
20580ggacccgttt tggtattcgt caggattctg ttctgttgga aaccgaacta aaatgagcat
20640gggcaaaaag agacgttttc agctcaagtg ttaagtccag aggtggacca gcttctggac
20700tgcggggctt agtctaggac agacattatg agagttttca ctctgcagaa accagaaaga
20760cagactctgc ccccacccat ggattcattt ctgcataatc tcttgcttca aaaatacaat
20820aagacaaatt ctccatgcca aaggatgcag tagagggtct ttttacagca acagaaatgc
20880tgtaggatga accccctata tggagtgagg gtgacggtgg gatagagatc ctggatatcc
20940aggcaaagaa ctgggttgga aaaggaagcc actaatagaa tagcgagaaa gaacactgta
21000aatgctgtga gccacagcta ctctggatac agtgtcacaa gagtgcactg gaatgcattg
21060aggctgacaa gccagggacc aggaagactg ctggtgaata atctttctgg ttttctgtaa
21120ggaacttgga aagcttatcc agagaggtgt gaaaacagca gaaaaaacac tgggacaaag
21180aaacggggca aaagcaaaca tgttccagat tagccgctga gaaacctaag aaacaaaacc
21240aggttttgtc ccagagaaaa gggagacttg ggcatcccct tggaggcccg gaagcttctt
21300gtttgtcaag tggattaaag ggtatttggt caaggattgt gtgtttacag cttaaagcaa
21360ctatgcacag aacccaggaa ctacctgcct ctcacgcttt caatggattg tcccagagac
21420tgctcccttt tgtgaagctg atgtttgtct tgtgttgaat gtaacacagt cacaaggagg
21480ctaccgtgac agggagcttg tattttcttt ctggatgtgg tattcaaagt gttgaacaac
21540cagtatggct caaccactga cccttcagtt cagctgccag aagtcagtct gggggaccct
21600actgtgcgag gctctattcc ttctgtttct ggatgatgtt gaggtctgga ggggccagga
21660tccaacatgc tgagtggggg cactcaggag tgttgggcag cagccacgta cccaccagta
21720tatctgaact gatctatacc agcatcacag gcccttattc tcctcataga gcacatgcta
21780agtaaatgca gccactgaat cagacagaag aacgccagcc agcaaccttc tgtccttacc
21840atgagcagtg ccttccagaa ccaccccagg gtggagggct gagaggtggg aagggagctg
21900gagcctctca tgtccacaca tgcattacct cagggatttc ccagcaacac aacaaaacag
21960gccttctctc gcccacttta ccagtggggc attgaggttt caggatggaa acaactattc
22020agggttttca gatggtgaga aagagtgaga atttaaaccc aggcctgaga tgaagcaggg
22080acctgtgcca ccaccccgaa cgccaccatc aagcatggaa ataaaggaaa atcttgagtg
22140ccttcaaggg aaatttcagg catccagcta gccctgagaa gtaagtaagt aacttgttag
22200gcaggaaggt aatagtagcc caaaacaata gccaaggaag ttagagtccg gagatgttta
22260gttttaggaa ctaaagataa catcttaaca cacgtccttg agtttgtcat tcagaaacct
22320ggacctccat gtggaccccc accaaacgga tctgctggcc tgcagacctc agatagaggg
22380agctgaggac tgcactctga ccaccttctt tcattctaaa tttcttccgg aggctcctga
22440aagccacacc catgagctaa agctaatatt cttttctgtt aaccctgaat tttcaggcaa
22500ggcatcgcct cctgaaccaa ctacaaatca aagaatctct gaatccacct atgacctgta
22560agcccctgct tcaagatatc tcaccttttt taggccaaac caatgtgtac ccttcatgta
22620ctgatttaca attttatcta acttctgctg ctttcctgaa aatttacccc gcctttaaaa
22680acccttacct gtgtgaaagg aaactatgtt ggccctccca aatcactaag ttaaagagaa
22740aattcaagct gggaactgct tagggcaaat ctgcctcgca ttctattcaa agtcatccct
22800ctgctcactg agataaatgc atatctgatt gcctcctttg gaaaggctaa tcagaaactc
22860aaaagaatgc aaccgtttgt ctctcaccta tctgtgatct ggaagcccac tgcctgcttc
22920aagttgtcct gcctttccag accaaaccaa tgttcatttt acatatgttg attgatggct
22980catgtctccc taaaaggtat aaaaccaagc tgtgctctga ccaccttggg cacatgtcgt
23040cagaacctcc tgagggtgtg tcacgggggc atgtcctcaa ccttggcaaa ataaactttc
23100taaattgaca cttgtctcag attttcaggg ttcacatctg ccagccatca gagagttcag
23160gttttgagca tgcgctgcct ggtccccctt gctcagggct ctgtgaataa acaccttcct
23220ttctagtgct gcaacttcag tgtggatatc tggacttaac tgcactgggc aagccgaccc
23280caatttgtca agcacgtcca tgtgaagaaa ccaccaaaca ggctttgtat gaggaataaa
23340acttttaatt cacttgggtg caggtgggct gagtccgaaa agagtcagca aagggagatg
23400gggaagggat tgctttatag gagttgggta ggtaatggaa agttacagta aaacgtggtt
23460atctattgtt agcagaggag ggggtcacaa ggtacatagt ggggagatca taagactcat
23520tgtccagaag aagaatgtca caaagttggt tgatcagcta aggtagggca gggacaagtc
23580acaatggtaa aatgttgtaa ttttggttac tcagttaagg caggaactgg ctgttttact
23640ttgtgggttt tttggctgct ccagacttct tggttcctgc aggccatctg gacatatatg
23700tgcaggtcac aggggttata atagctgagc ttcagctcag aggcctgaca caattcagtt
23760atataatagt cctaatgacc caaaagccag tactctcttt tttttttttt tttttttttt
23820tttgagatgg agtctcgcgc tgtcgcccgg gctggagtgc agtggcgcta tctcagctca
23880ctgcaacctc cgcctcctgg gttcaagtga ttctcctgcc tcagcctcct gcgtagctgg
23940gattacaggt acatgccacc atgcccagct aatttctgta tttttttttt agtagagatg
24000gggtttcacc atgttggcca ggctggtcac aaactcctga cctcaaatga tctgccacct
24060cggcctccca aagtgctggg attacaggcg tgagccacca cgcccagcca agcccctgct
24120ctctttacag ccagggctgc ctccctcaca cacctgccca aagggctggc agcatctgca
24180ctcaagtgaa gagatctaag cacctttgag ccccagagca gcaggtgggg gctaccacca
24240gcatggggcc tttcatgcgg gagatcccac agcccttctc cttccccttc ctcctccctt
24300gacaatgtgt tcaggtctcc cacagccttt ggcaccaaga aattccaata gatgttaacc
24360agcctggtaa tttagtacag cgttagcacc aagtcgccaa ggttgaggat atgctaccgt
24420gacacaccct caggaggttc tgacaacatg tgcccaaggt agtcagagca cagcttggtt
24480ttataccttt tagggagaca tgagccatca ctcaacatat gtaaaatgaa cactggttca
24540gtctggaaag gcaggacaat gtgaagcagg cagggggctt ccagatcaca tatgtgagag
24600acaaacggtg ctatttccaa atatttccaa actactctct tttccagcta ctcaccagga
24660ggaaagtaaa tgcctgtcag acatggttac taatcaccgc cggtgggtgg gatgaaaagg
24720agcttactga tgactggcca ggtgctcacc ctcaccaggt ctcagattcc tctttggcta
24780atgagggagt caattctcta ttgggggtag caagttcaaa tgctcggggc aagtcaggta
24840acacaaccca gtgaagcagg tatgatgttt gtatatgaag tacttatgag cctcactttc
24900ctaagtaaat aggctttctc ctattttcct tgaaatgcat accactgcca gtctgtttct
24960ctttatccca attttgacag agacatatgt ataaaaaata catttttagg ccaggagtag
25020tggcttatac ctataatccc agaactttgg gaggcccagg taggaggatt gcttgaggcc
25080agaaattcaa gagcagcctg gacaacacag tgagaactcc atctctacaa aataaaaaaa
25140ttagccaaat tggaggcaaa ggcgggtgga tggcctgagg tcaggagttc aaaaccagcc
25200tgatcaacat ggtgaaaccc catcactact aaaaatacaa aattaggcag ggcatggtgg
25260ctcataactg taatcccagc acgttgggag gccgagacgg gtggatcatg aggtcaggag
25320atcgagatca tcctggctaa cgtggtgaaa ccccgtctct actaaaaata caaaaaaagt
25380taggtgtggt cgcgggtgcc tgtagtccca gctattcagg aggctgaagt aggagaatgg
25440cgtgaacccg ggaggcggag cttgcagtga gccaagatca cgttactgca ctcccgcctg
25500ggtgacagag caagactttg tctcaaaaaa aaaaaaaaaa aaaaaattag ccaggtctgg
25560tggtgcatac ctgtaatccc agctacttgg gaggcaggag catcacctga acctgggagg
25620tggaggttgc aatgggccga gatcatgcca ttgcactccc gcctgggcaa caagagcaaa
25680actccacctc aaaaagacag gacaggacac gacacgacac aataagacga gaagaaatat
25740tatgtctttc acaaaaatct ttcagaaaat aaaaaaggat agatcatgtt ccaactcctt
25800taatgaggcc agtgttaccc caatatcaaa gctaaacaaa cacaccaaaa taaacaaaaa
25860ttatagaaca atatctctca tgaatagaga tgcaaacata ctcaataaga tattagcaaa
25920caaaaatcca tcaacatgta caaaagatta tacaccatga ccaagtatga tttatcccag
25980aaatgcaagg ttggtgtgac atccaaaaat caagtaatgc tttctctagt atatggtgag
26040gtttttaaaa ttaattaatt aaaaacaaca aaacctcaaa atcagtgaat gtaatgtact
26100agattaatag actaaaatac atcacattgt catttcaata gatacagaag catttgacaa
26160tagccaacat acattcgtgg taaaaactct caacaaacta ggaataaaag taaacttctt
26220caatctgata gagggcaact acagacaacc tacagttaat gtacttaatg atgaaagaca
26280gaatgtttcc cactcaagat taggaaacag gcaagaatgt ctactcttac cacttccatt
26340caacattgta ctactggtca tagccagggc aacttaaaaa gggggtagag ggagaaagag
26400agagaaaggt aaattaaaga catctagaat gaaaagaaaa aagtaaaact gtgtttattt
26460gtagaccaca tgatctgtat gcagaaaatc ccaagtaatc tatcaaaaaa ttttactgga
26520attaataaat gagtttagca aacaaggttc aagtatataa ggccgatatg taaaaatcaa
26580ttgtatttct acatactagt aataaacaat gtgaaaatga aattaaggaa acaaaatttc
26640attcacagta acacctaaaa gaataaaata cttagaaatg agtttaatga aagaaatgca
26700agacttacaa taaaaactat aaaactttgc tcagagaaat tgaagaagtt ctaaattaga
26760ggcagcaaac tacagtccat ggccctgaac tggccccctg tttgttttgt aaataaagtt
26820ttattgaaac acaatttttt tttttttgag atggagtttt gctcttgttg cccaagctgg
26880agtgcaatgg tgtgatctca gctcactgca acatccacct cccaggttca agcgattctc
26940ctgtctcagc ctcccgaata gttggaatta caggcgtgcg ccactatgcc tggctatttt
27000ttttgtattt ttagtagtaa cggggtttct ccatgttagc cagctggtct tgaactcctg
27060accttaggta gtccacccgc ctcagcctcc caaagtgctg ggattacaag catgagccac
27120cgcgccgggc caacactgct tttacactac aaagcaaagt tgaatatagc ccacaatacc
27180acaaatattt ttactatctg gttctttgca gaaaatttgc taatccctga tttaaataat
27240cccatgttca tagattaaaa gactctattg ttaagatgac agtcctcccc caatttattg
27300ataaatttct gccaatttaa aaaaaattaa tgtcgttcct aagaaagtga gtccattcca
27360aatactgcga aatcaaggga aaaaattaaa aagtcagtcc tggctgggtg cagtggctca
27420cgcctgtaat cccagcactt gggaggccga ggcaggtgga tcacctgagt tcaggtgttc
27480aagaccagcc tggccaacat ggtgaaacag tgactctact aaaaatacaa aaattatctg
27540ggcatggtgg catctgcctg taatcccagc tactagggag gctgaggcag gagaatcgct
27600tgaacttggg aggcggaggt tgcagtgagc caagattgtg ccaccacact ccagcctgga
27660tgacagagtg agacttggtc tcaaaaacaa aaacaaaaaa aatagtccag caggtttttg
27720tggaaattga taagcttttg ctaaaattta tatagaaaag cgaatgacct aggatagcct
27780aaatattctg aaaaagaaca aagctaaaaa aaaaacttac actatccaat ttcaacactt
27840gctattgtta aagaaaaatt ttgcaccaga cacttgtcaa aaatgtcaag acagatttta
27900ttcagactac tgtggcaggg aagagacact tcagcattaa ctgagctcaa ttctaccaaa
27960ccagaaggca agaggctttt taaatgctag gatgtgttaa gggaaaagta ctgaaggatg
28020ctagttgagg ggagggaggt ttggtcaatg agtacactga ccagattgtg aacttgtgct
28080tatcaaattg tgaactggtg cttatcaata tcagggtcct accctaccac caagattggg
28140agacagaggc tctgtcctac ctaatgatta catttcgaaa ggatggctcc caggtccttg
28200ggaaagacat tcctgtgtta tagaagatac atttccaaga gacagagaaa ggatttctaa
28260ctgccagttt tctaaagtaa atcctttagg aaaagggggt tcgggggcct atattcaggt
28320tttgcctgga acaaatcata aattctttgg gcagcattga attttattag gcagaaactt
28380aagggggctg aagtcatcat cctaaggatg tggccatgaa ctgttagaaa caacagtgtt
28440tgttgaagtc tcttagtgcc agaggtggat gaaatcattt gtgctgagag tctgcagttt
28500gcttttttat ttatatttta ttttttatta tttatctttt tttttttttt tttgagacag
28560ggttttactc ttgttgccca ggctggaccc aggctggagt gcagtggcgc catctcggct
28620cactgcaagc tctgcctccc gggttcacgc cattctcctg cctcagcctc ctgagtagct
28680gggactacag gcgcctgcca ccacgcccgg ctaatttttt ttgtattttt agtagagacg
28740gggtttcacc acgttaatca ggatggtctc gatctcctga cctcatgatc cacccacctc
28800ggcctcccaa agtgctggga ttacaggcat gagccactgc gcccggccca tttttttttt
28860ttttttaagt ataacatatc tctattaagt tgaatagaaa cactaacaaa gactagtatg
28920catagtgatt atagatgtag acagataaaa aagaaaaagg gatacaaggg cgcattgaat
28980ttatgacaat attgtctcag taccacagca ggagccaact caccccaggg ctggcttccc
29040aagaccagct ccgggaagcc tgatctgctc agccttgatt actggttgac cgggactggg
29100gatccagctg gcaggacagg ctgtagctct caccctcact gggctgctct ctgacccaga
29160accaggcccc cagttcctcc ccaggatgaa ggctgtaatg atttgcagcc acctgagcag
29220ctgggtctcc ttttctggga gtccatcata ttctcatcca cattaccatt catcgcagtg
29280tccagcatca ccaggtcggt gaggaatgtg ccaaggacag ggacaattcc cttcttctgc
29340tgctgccacc ttgcatgggc tctgtggggg cttgcctcca gggtggccga cttggaggtc
29400cccacactga tgagcagatc cctgttcggt ggcttgtctt tgctgcacag ctttggacat
29460ttttctgttg ctttatcttg gccactcaga gcagagcagc cacaaccccc caccccacag
29520tgcctgggcc aaagtggccc aggttagggt ccttgaaaga cgagacacca gagagttccc
29580tctccttctc tttccctctg tcccccacac cctctttcca ggagcacaaa gaggtcatgt
29640gagcacgcag tgggagggtg gccacatgtg agccaagaga ggggcctcag aatgaagcct
29700gcctttcctg caccttgatc gtggacttcc agcccccaga actgtgagaa atgaatttct
29760gttacttaag cctcccagtc tgtggtattt tgttatggca gcctgagcca actaatacaa
29820caggaaagaa acattccaat ctccgtaaat tcatttttaa gttcattaat gtttaaagtg
29880attaacagtg ggtacacttt gtaaaccatc aagatctcat acagtcatat tttatgattc
29940cccataatta gttggtcatg gtttgtaact cctgacaaac attgttttta gttttagag
29999830000DNAHomo sapiens 8aaaaaaaaaa aaaaattgag taaataaata aatggtagaa
tttagatttt tttttttttt 60tttttttttt ttgagatagg gtttgatttt gtcgtttagg
cgtagtggcg taatttcggt 120ttgttgtaat ttttgttttt tgtgtttaag cgattttttt
gttttaattt gttgagtagt 180tgggattata gatatgcgtt atgatgttcg gtttttaaat
ttttttgtag agatagggtt 240ttgttgtgtt atttagattg gttttaaagt tttgggtata
agcgattttt ttatttgatt 300taggattata agtatgagtt attacgttta gtttatagtt
tagattataa tttatattat 360ttatttgtga ttagatgtta aatagtattt ttttttgttt
tagttttgat agaggttttt 420tatatttagt tgttaattgt ttttttatat ttcgtagtaa
aggaggtgat tatagtaggg 480ttttatggat tttatttaag agtgattttg ttataagggg
taaggtaatt ttattagaaa 540ataataggag atatagatta cgatgtagaa gttaagataa
tagttatagg ttgtgagtgg 600aaggggagtt gtggagagag aagtttataa aagttttatg
aggaagttag tttagaatat 660gattttaggt agaaggagtt tttaattttt ttgtttttta
ttttttttta atttagatgt 720agtttttgga atgagatttt gtgtaattta aaatgtaatt
tagattattt ttttaaatta 780tttgaaggtg aataagaaag ttaagggatt tgtttagatt
gtttaattgg tttatatagt 840atgtttaaat agatagggga taggggaaaa taaagttttt
tattagttgt ttattgattg 900tttaatagat gattgtttgt ttatttttga tttgttacgt
atatatagtt gagtttaagt 960tttaggagaa tagggttttt ttttgaaatt tttttggttt
attgttgtat ttttaggttt 1020agagtattgt ttggtttagg tgtttagtat agatttgttg
agtgaataga tgttttttaa 1080tatagaaata gttaaaagag aattaatgtt gtcgttgttt
taagttatta agttttcggt 1140ggtttgtgtg tagtaagagg tagttgatgt tggggttttg
tatttaggtt attttttatt 1200tgtaaggtag agtttttggt ttttaatttt tttcgggttg
tatgtggttt tgggggtatt 1260ttttttaggt ttgttttaag ttgttttata ggtttgtttt
aagttgtttt ttgttttaag 1320ttgttttgat tttaagtaaa tatagaggaa tgtgggggat
atattataga agatattttt 1380tgatgaagag gaaggaggga taaataggaa tatcgtaaag
aaaagaaggg atttaatttt 1440agtttgaaga tatatatttt ttttatcgtt tattagatag
taggatacgg gtagtagagt 1500gtagatatta gaaatagtaa gagtggtagt ggttttttga
gtaagggatg tataatgatt 1560tattatagag tttaggaaaa atagaatttt tatttatatg
tattttgttt tgttttgttt 1620tgtttgggtt tgggtttgtt tgtttgtttg tttgtttttt
gagacggagt tttattttgt 1680tatttaggtt ggagtgtatt gatgtgattt tggtttattg
taattttcgt ttttcgggtt 1740taagtgattt ttttgtttta gtttttcgag tagttgggat
tataggtgtt tgttattacg 1800ttaattttgt attttagtgg agacggggtt ttattatgtt
ggttaggttg ttttcgattt 1860tttgatttta aatgattcgt ttgttttggt tttttaaagt
gttgggatta taggtatgag 1920ttattgtgtt cggttattta tatgtagttt tgtttttttt
tttaaatgtt ttgtattttt 1980tataatgtgt attaaattaa tgtatatata aattataaat
taaaaggata tatatattta 2040agtgtttgtt taaaaatttt tttattgata ggtatggtta
tataattatt gattattaga 2100ggttagagga gaatgaggtt tggtttggga gttttgtgtt
tattagaagt atattagatt 2160atttatttat tgatagaata ggtttttttt gggttttttt
tttttattac gatatatttg 2220tagttttttt ttttgaagat tttttggtag ttgtttttgt
tataatttat aggtgtagaa 2280atattgttat tgagataaaa ttggggtttg ttagaagaga
aaggatttcg agtatttttt 2340ttttagtgtt agttttgttt aggaagttat ttatatttta
atttaagggt attttttggt 2400tggtagtttt agagtgtgat ttttaggaat ttggattagt
ttgtattata gagttatata 2460gtttattttt atattttatt ttgtaggtat agaaggttat
tagtaggtgt tgataggttt 2520aggggtaagt ttggttattt tagaaagtgg tagtttgttt
tatgttggtt ttttttattt 2580tttttgttta ggaagtggtt agtatattgt tgttagaatg
tggaaaggaa ataaatttta 2640gttttgattt ggaggtttgt tggtaaagga gtttttttaa
ttaattagag taatgagtta 2700ttttaggtta gtgttatgga gtttagggag aagttgatga
aagttagaga ttatttttaa 2760ttgagttgta gtatatataa atattttgaa gagttttgat
gagatgagat gatattaatg 2820ttagaatatt tatgtattat tattattatt attttaattt
tttgagatga attttatttt 2880gttatttagg atggagtgta gtggtacgat tttagtttat
tttagttttc gttttttagg 2940tttaagcgat ttttttgttt tagttttttg agtagttggg
attataggag tatattatta 3000tatttagtta atttttatat ttttagtaga gatggcgggt
cggcgcgggg gaggttttat 3060tatgttggtt aggtaggttt cgaatttttg attttaaatg
atttatttat tttgtttttt 3120taaagtgttg ggattatagg cgtgagatat tatgtttagt
ttagtgttag aatattaagt 3180ggaaaaatta gtttataaaa ttgtgatata aaatttgata
tttattatgt aaaagaaaaa 3240atatatagaa aagagatata aagaaatgat gatagggtcg
ggcgttgtgt tttacgtttg 3300taattttagt attttgggag gtcgaggcgg gcggattacg
aggttaggag atcgagatta 3360ttttggttaa atcggtgaaa tttcgttttt attaaaaata
taaaaaatta gtcgggcgtg 3420gttgtaggcg tttgtagttt tagttattta ggaggttgag
ataggagaat ggcgtgaatt 3480taggaggcgg agtttgtagt gagtagagat tatattattg
tattttattt tgggcgatag 3540agtaagattt tattttaaaa aaaaaaaaaa gaaaataaat
gatgatagaa gtttaggtat 3600ggtggtatat gtttgtaatt ttagttattt ggggttgagg
tgagataata gtttgatttt 3660aggagtttga gattagtttg ggtaatatag tgagattttg
ttttaatttt tttaatataa 3720ataaataaat aaataaaaat agaatgttaa tagttattat
ttttatatag ttgggattat 3780gagtaggtat tttttttttt tttttttatg ttttttaatg
attttataat ttatatgttt 3840atttagaaaa ttgggaaata ttatttaaaa ttttttatgt
tttttatgtt taaaggtgat 3900tgttggttgg taggaatgta aaatggtgta gttgttgtgg
aaaatagttt ggtagttttt 3960taataagtta aataaaaaat tattatatga tttagaaagt
tatttttggg tatatatttt 4020aaaagaattg aaagtaggga tttaaatatt atatgttaat
gtttgtatat tgataaatat 4080tgtatgttaa tgtttagaat aatattattt attttattta
ataggtagaa gtaatttaag 4140tgtttaataa ataaatggat aagtaaaatg tggtatgatt
atgttatgga atattatttt 4200ggtagaaata agaatgaagt attgatatat gttataatat
gaatgaattt tgataatatg 4260ttgagtgaaa taagttagat acgaaaagat aaatatagtg
tgattgtatt tatgtgaaat 4320atttagagta ggtaaattta tagagataga aagtagattt
gagattatta taggtttggg 4380ggatagggaa tttgagttat tgtttaatgg atatagagtt
ttagattgga gttattaaaa 4440agatttggaa atagtgttta tggttgtata gtattgtgaa
tgtatttaat gtcgttgaat 4500tgtatgtttt aaaatggtta tattggtata ttttatgtag
tatatatttt attataataa 4560aaaaaaaagg ttaaaaataa agtggtgttt gttgaagaga
tataattttt ttagtttatg 4620gtaggaagtg aaatggtgaa tttgtgttgt ttgtgtgtaa
atataagttg gtaaggtaaa 4680aggtatatta ttgtatgttt attttaatgt aaattagatt
tagttttata gaggaaatag 4740tagagagaga gagggaggga gaaattgtta atatcgggta
tttagttaat tagagttagt 4800ggttggatga ttttggtttt ttgtttttta tttttttttt
tttatttaag ttagggagtt 4860agttttgggt tcgggtagcg tggagtttag gatatttttt
atgttttatt tgggagtttt 4920tgtttgggtt tttagtttta ggttttattg ttatcgagta
ttttgagatt tgggtagcgt 4980tttatagagg gtgtagtggg tttaaaaatt ttttttagaa
aaagtttaat taaagtttgt 5040atttttttat ggaggagaaa aaagtttatg agaaaaataa
aggaggtgag gtaattttag 5100ggaagaaggt gttgatttat tttttttgta tggagtagtg
tgttaaatgg aataggtaat 5160atggtagtag aaattttgaa attttgaaat ttgattaggt
gttagttatt gttttaggat 5220tataggatat agaattggta tagagaaggt attaagtgtt
tgttgggtta aagaatatat 5280atatagaatt ttatttaatt attatagtta tataattata
atgtgaggaa atagaggttt 5340agagatattt agaaattgat ttagggttat atagttagta
tataagggaa tggagattta 5400gatttaagtt attttgattt taatagtgag gttttttttt
ttttattgta aggagtagtt 5460tggttcgttt ttatattagg attacgagaa ttttagttta
tagtggagaa ttttagaaga 5520gggtagggat gttaaatttg ggtgatatgg atattttagt
tattagggag ttacgttttt 5580atgtgaggga gggattagtt attagttttt tatttggttg
ggatataatt atattttatt 5640agagtgatat taatatttga tataaagata atgttaaatt
tttggagtta taaaattaag 5700gtttagttgt aatatttagt gatttggtta atgcgatgtt
taggagtttg ttttgagtag 5760ttagttattt tttagtttaa tatttttttg ttttattagg
tatttagtat gaggtttgtt 5820cgataaatgt ggttggaatt ttttaggtat tttaagttta
gtaggattta ggtgttttgt 5880tttaagttaa gtgtttgttt ttttattcgt agtgttttta
taagattttc gaggtatttg 5940ttaaaattta gattttttgg tttttttttt atagattttg
attaattagt tttgggatgg 6000agtttaggaa tttgtatttt attaagtttt ttaggtgagt
ttgtgattag ataagtttgc 6060gaaatgtttt ttgatattaa tgttttgtta ttagatttat
ttttgttttt gtagaatatt 6120ataagttaag gttatatgaa gaggttagat ggatttgggg
ggttgggagt taggtagttg 6180ttatggatat agttgttgag gaaataggta aattaggacg
gtattgcggg tattttgtgt 6240ttcgtttgtt taagttttag atttttggtg tttatagtaa
tttagtagaa aatatttgta 6300tagtttttgg gttgacgtga gttaaatttg agtagtgaag
tagtataagt tttatggtgt 6360aaggtatatt atagattttt ttttattttt tatatttgat
ttttttgttt atgtttagag 6420ttattgttat ttttataaat ttataaggtt tagattgagg
tgatgatcgg gttaattttt 6480ggaatagtgt tagataatta gatatttttt tttgttttta
ggagtttatt tggaataaag 6540ttatttatta agttttaaaa ataatagaaa agaaatgaga
ttgaggtgga aaataattta 6600agtgtttatt aatagatgaa tggagatgta aaatgtggtt
tttttatatt atagaatatg 6660atttagtttc gaataagaag gtaatgttga tgtaagcgtt
aatatagatg aattatgagg 6720atattatgtt aagtgaaatg agttagtttt aaaaaagtaa
ttattgtatg attttattta 6780tatgagggat ttagagtagt taaaattata gagatagaag
tgaaatggtg gttgttaggg 6840gttggggaga aggggatgga gagtgattgt ttaatgggta
tagagtttta gttttgtagg 6900atgaataagt tttggaggtg gatggtagtg atgttataat
aatgtgaatg gatttaatgt 6960tattaaagta tatatttaaa attgttaata tggtatattt
tacgttacgt atattttatc 7020gtaatttttt ttttttttag atatagtttc gttttgtcgt
ttaggttgga gtgtagtggt 7080gcgatttcgg tttattgtaa gttttgtttt tcgggtttac
gttatttttt tgttttagtt 7140ttttaagtag ttgggattat aggtattcgt tattaagttc
ggttaatttt tttggtattt 7200ttagtagaga cggggtttta cgtgttagtt aggagggttt
cgattttttg atttcgtgat 7260tcgttcgttt cggtttttta aaatgttggg attataggcg
tgagttattg tgattggttt 7320ataatttttt aaaaagaaag aaataggatt gaaaattaat
tttttaatat tataatatta 7380agaggaagtt ttaatataaa ttatggattt aggtgataat
gatgtgtaag gtaggtttat 7440tagttataat aaatatatta ttgtggtgga cgatgttgat
aacggggagg tcgtgtatgt 7500gtgagggtag gaggtatatg ggaaattttt gtatttttgt
ttagttttgt tgtgaattta 7560aaattatttt atataaagtt tataaaaaat taaaataaaa
attaaatttt tgaagattgg 7620aattttgtaa gacggagtag ttgtttttta gagaatagtt
atgttttttt tttggttagt 7680attggtttga gatttatagg gtgtaatatg aaatttttgt
ttcgtagtaa gtgtatgttt 7740tatagttgtt agtttgttat ttcgagttta ttgggtgttt
gttttttttg agatttatgt 7800attttaaaat aatatatata atattattag tgtaagttat
gtaatgagaa taaggtttat 7860taggatattg agttttgttt ttattattta tatttttttt
agagagagag gagtttaatt 7920attttaaagg tatttaagaa tggtgttttt gtatttattt
acggttaatt ttagtattgt 7980ttagtttatg ttattgataa tgttttttta tagaaagttt
atattttttt aagttttatt 8040ttagaataga tattttaaga aaaaataagg tttttttatt
gaattagata gttatgtgag 8100tttgtgggta tttgggatta tatttttttt tattggatta
tttttggatg agaattttat 8160ttttatattt ttttttgttt tgtatcggtg gtttttaatt
ttgggtgtat atgtgaaata 8220ttttgggtat ttgaaagtta tttatgtttt agttttattt
tagaataatg aaattagtat 8280ttttgggtat agggtttagg ttattgtgtt tttagaaggt
tgtttaagtg aggttaggta 8340tagtggttta tatttgtaat tttaatattt tgggaggttg
tggcgggtag atgatttgag 8400gttaggagtt cgagattagt ttggttaacg tggtaaagtt
ttatttttat taaaaatata 8460aaaattagtt aggtatgatg gtatatattt gtagttttag
ttatttggga ggttgaggag 8520gtagaattat tggaattcgg gaggaggagt ttgtagtgag
ttgaaatggt gttattgtat 8580tttagtttgg gagatagagt atgattattt taaaaaataa
agaaataaat aaatagaaat 8640ataaaggttt ttatttgtag ataggtttat ttattaggtt
gtaataatat aataaatatt 8700gaaaagttaa gatttatttt tggttgaatt ttttggaatt
tttttattag gtatgaaata 8760gtatgatgta ttgtataaag ttacgaaggt ggtaaagatt
ataacgttgt ttaggagaat 8820attttgtaat gtgaagaaag gttagggtgg agaatgaatt
aatttaaagg ataatttttg 8880attgagtgtt gtgtattagg atttcggttg ggttttgttg
agaggttatg atgtggagtt 8940tataattgaa taaattagtt aatttaggtt gaaattgtag
aataaggaaa agtatttagt 9000tgattttgtt atttttttga gatgagttaa ataaataaat
aaattttaat atggtaattt 9060ttgggaattt ttgaaaggga agatggtgtt aaaaaaaatg
ttgaggaggt tatttgtttg 9120gattgagttg tattgggttt aatagattaa attaaaatgt
agtcgtttat gttgaagttt 9180tttgttatta agttaaaatt aagttgttta attaattttt
taagaaatta gaagagagtg 9240agaggtaata gttaaatgtt taataggtta gttttagttg
atatgatgag aaagtttttt 9300ttgttttaat ttataagtaa agttatttcg aaaagattaa
tttgtttttt gttttttgtt 9360tttgtttttt ttggtttttt tttgtttata aagttaaatt
tttttgttag gtacggtggt 9420ttacgtttgt gattttagta ttttgggagg ttaaggcggg
tggattatga ggttaggaga 9480tcgagattat tttggttaat acggtgaaat ttcgttttta
ttaaaaatat aaaaaattag 9540tcgggtgtgg tggtgggtgt ttgtagtttt agttattcgg
gagtttgagg taggagaatg 9600gtacgaattt aggaggcgga ttttgtagtg agtcgagatt
tggttattgt attttagttt 9660gggtgataga gcgagatttc gttttaaaaa aaaaaaaagt
taaatttttt tggttagttt 9720tgtagaatat tcgatttatt ttatggaatg aagtgttttt
tgattataga tttataagta 9780aaagttaatt gagggttagg cgtggtggtt tatgtttgta
attttagtat tttgggaggc 9840ggaggtgggt ggattgtttg aatttaggta attgaaatta
ttttgggtaa tatgatgaaa 9900tttatttttt attaaaatat taaaaaatta gttgggcgtg
gcggtatacg ttagtaattt 9960taggtattcg ggaggttgag gtgggagaat tatttgagtt
tgggaggcgg aggttgtagt 10020gagttaagat tgtgttatcg tattttagtt tgggtaagag
agtgagattt cgttttaaaa 10080aaaaaaaaaa aaaaaaaatt aattaagatt tttaaattaa
atttgttgta attttgtttt 10140ttgataatgg gaatgaaggt atttattttg taagattgtt
aaaattaatt atgaagtgac 10200gttatttgaa gtagtatgtt atttatatat gaatagaaag
atagggaggg tatagtgttt 10260taattttgta attttagtaa tttgggaagt tgaggtaggt
atatcgtttg agtttaggag 10320tttaagatta gtttaggtaa tatggtgaaa atttattttt
ataaaaagta taaaagttag 10380gtgggtatgg tggtgtgttt ttgtagtttt tgttatttgg
gaggttgagg tggagaatta 10440tttgagttta ggaaggttga ggttgtagtg agttatgata
gtattattgt attttagttt 10500gggtaataga gtgaaatttt gtttttaaaa aaaaaaaggt
aaatatagag attaaaatag 10560aaaatttaga aaattttaaa ggtagttgga tagggtaaaa
aagaaaaaaa taaaatttag 10620aaagtataaa tatatgtaga agtttattat aaaataaata
tgttttttga gatttgtgga 10680gagcgattat ttaataaatg ttattagaat aagtggtgaa
ttatttgggg aagaaaaaaa 10740gttaattttt tatttgagtt tttattagtg aaataaattt
taaaaggatt taaggtttaa 10800gtttttataa taagattata aaataattat agtatatata
tataaatatt tattttaagt 10860ataaaagaaa aatagatttg agaatataaa cgttataaga
atataattga aattagaaag 10920atattaaatt tttagaataa tattattata tattaatggg
agaagaaaaa tattttaaat 10980agaaaaaggg taagagaaat aaagtggtat tttatatata
gatatatata tatagaaatg 11040atgaataaat atatgaaaaa gtttaattta ttgataataa
aagaaatgta aatatgtagt 11100atgaaataga agtataatgg gatggtgaga ataattattt
aatttataat atttagaggg 11160aaatagaggt atttatggga ggtgatatgt ataagaatga
tttattttga ataatcgtta 11220aggagaataa tttgttatta tatatttata tttttaaaat
cgtttataat atttgatttt 11280atttttttat ttttaagatg tgattttaaa aatggtatag
agatagagat agagatagat 11340ttttttataa cgatgtttat tgtagtggaa aaatggaaag
aaataaaatg tttaagaata 11400gagttagtgg agaatagtga tttagaaaaa tatttggtga
gttagtaagg tagaaggtga 11460agttatatga gtaattgatt attatatgtt acgatatgaa
atgaagtagg ttatgaaata 11520tatgatgcgg agagagatgt ttagaatata ttgagggttt
tgtattaaat aataagttat 11580ttttggatgg ggagattatg ataatttttt tttttggatt
ttttttttta ttttttaaat 11640tttttataat aaaatatatg agttttatag ttataaagaa
agaaagattt tttaaaaatg 11700atataacgtt ttgatatatt tagtttaagt taggatttgg
agtggtattt gttttgtttt 11760atttttttga gtagttttta gttcgaaatt attgtggaaa
tttaaatttt tttttttaaa 11820aagttagttt tgttgatagt atttttattt tttttatgta
gaaagagggt ttttggggta 11880ttggggatta ttggaatttt ggggatttgg ttttattgag
ttataaagta ggagagttaa 11940tagggataag tagggttgta ttttaggata agtgatatgt
gtaaaatttt taagtttttt 12000gtagttttag agtagaggta ggagattgaa ttattggggg
agagtttaaa tatggtgata 12060gtagagagta aattatttaa ttttgagatt atttatttga
tagtatttta gaaatagttt 12120aatgttgttt tttttttttt tttttttttt tttttttttt
ttttttgttt tttttttttt 12180ttttttttga aatttagttt tttttttttg taaaaagagg
ggttttttta tttgggtgat 12240tattagagtt atttagggag tttataggaa atataattta
ggagttagtg ttttagtgtt 12300tttatttagt tttttaggtg agaatttagg aataatgatt
aatttagtgt tgttttaatg 12360tgtatataaa tttttgggag ttttttttgt tttgttttgt
ttgtttgttt gtttgttttt 12420ttttgggacg gaatttcgtt ttatcgttta gagtagagtt
tagtgatgta attttggttt 12480attgtaattt tcgtttttcg ggtttaagtg atttttttgt
tttagttttt tgagtagttg 12540ggattatagg agtgtattat tatatttggt taatttttgt
atttgtagta gagacgtagt 12600tttattatgt tggttaggtt ggttttaaat atttgatttt
aaatgattcg ttcgtttcgg 12660ttttttaaag tgttgggatt atgggcgtga gttatcgcgt
ttaattagat agtttgttaa 12720ttaatttgta gattttgttt tagtaggttt ttagatttat
acggaagttt ttaggtgatg 12780tttacgttgt tggtttgagg tttatttttg agtagagagg
ttttaaagtt ttttttattt 12840ttgtttttta ttttgttatt agtttttttt ttttttgtgt
tttttttttt tgtatttagt 12900aattttgagt ttttgatgtg tgtaggagat tatattacga
cgttagaatg ggttaggatt 12960ttttagatgg tatagcgagt tttgaggtga agtaggttgg
tagtattttt agaagttgtg 13020ttttttggtt gggcgcggtg gtttacgttt gtaattttag
tattttggga ggtcgaggcg 13080ggtggattat gaggttagga gatcgagatt attttggtta
atatggtgaa atttcgtttt 13140tattaaaaat ataaaaaatt agttgggcgt ggtggcgggc
gtttgtagtt ttagttattt 13200aggaggttga ggtaggagaa tggcgtgaat tcgggaggtg
gagtttgtag tgagtcgaga 13260ttgcgttatt gtattttagt ttgggcgata gagagatttc
gttttagaaa aaaaaaaaaa 13320aaaaaaaaaa aaaaagttgt atttttttag gtaaattatt
gtttttggaa gggttatttt 13380atgattaaga gttgggggtt tggttaggta taatttttta
tatttgtaat tttagtattt 13440tgggaagtta aggtgggagg attatttggg tttaagagtt
agagattagt ttgggtaata 13500tagtaaaaat ttatttttat tataaataat aataataaaa
taaataaata aataaataaa 13560gagttggggg tttttaggat tttaggtagg tagaggtatt
gttagttttg tagtttgtta 13620agtttattgt gaggttgtat agagtatagg ttggaggagt
ttgtttagtg gagatgggga 13680ggatttttta ttttattgtt gttttattat ttttagtttt
gttttcggat agtttttgag 13740agtaagtttg ttttgtcgag tggtttagag gaggttcggg
gttttttttt gaggattttt 13800ttttattttt tttggttttt tttatttggt tggttgtttt
ttgttttatt ttttttgtag 13860tttttagagt agtttagttt agtttttagc gttagtaaag
aatgttttaa gaaagattaa 13920gtttaagata taagaaaagt ttgtatgggt ttttataaat
tttgaagtaa aatttttttt 13980tggttaggta tagtggttta tattagtaat tttattattt
tgggaggttg aggtgggtag 14040attgtatgag tttaggagtt taagattagt ttgggtagta
tagtaaaatt ttatttttaa 14100aataaatata aaaattagtc gggtatggtg gtattcgttt
gtggttttag ttatttggga 14160agttgagatg ggaggattat ttgagtttag gaaggtttag
gttgtagtga attatgattg 14220tattattgta ttttagtttg ggtaatagag tgagattttg
ttttaaaaat aaataaataa 14280attaataaat atagttttaa atttaagatt tttttttaat
atatgatata gtgaaaaggg 14340tttagggaaa tggtttttaa atttgagtat taaaatgatt
tgggtagttt tttaaattga 14400tgttttgttt ttattgtagt ttaattaaat tagagtgtcg
gggtggaaga tgggtgttag 14460tgttttttaa aattttttcg gagtttttat tgtatagtta
agtttgggag tttttggtag 14520ggagagagga atgtttgggt ttttatttta gtttatttat
aggtttgcgg tgagatttta 14580gtggtagttt tttttttttt ttaggtttag tttttttatt
ttaaaaacga tgtagttatg 14640tagttttgaa attattttta attttaaatt atttatatag
gatttaagaa ttagagatat 14700ttatgaaatt taattgaatt ttagaattat tggaaggaaa
aataatttag attttagaga 14760atagaattag tgttaaattt attataattt tcgtaagttt
ttagattttt atattttgta 14820tattgaattg taggtaataa tataaggttt ttaattaggt
tatatttatt atttaatgtt 14880atgtggtaga agtatacgta tattaaaata ggaatatata
tttttttttg tttttttagt 14940ttttatatat aaaattagtt atttattatt gtgataagta
ggatttttta tagataggta 15000tatttaggta aagttgtatt aaatttttat attggagagg
gtaattacgt attcgatatt 15060attgggaatg ataataaggt gagaatatat tgagtgttta
tagagggttg attattaaaa 15120ggaaagtatg gttttagatg atttatgagt tattaattaa
ttatattttt atttaagtta 15180tgtgtttatt ttgtttttat ttcgatttgt atttgttgtt
gatttaaaag agtagaggag 15240gagagaattt ttttttgttg agttttcgtt ttatgttggt
ggattgttag tacgtgatgt 15300gtttttattc gattttatat ataaagaaat agttttagcg
atgttagtta taatagttag 15360gtggtaaagg tgggtttttt gaaggtataa gaatagttta
taggttagtt tattaggttt 15420atcgggtttg ggtttgtggt ttagatgatt ttgtttttag
ggtttgatat gagaatagaa 15480atgggttaag ttattttgta gggtatagga gagtaagaat
tatatatagt tcggggtttt 15540ttgataagtt gaattgttgg atttttttag agtttttttt
gaatttgtag tttatttttt 15600gttgtattaa gtaagttttg tttttttttg ttttatagtg
gttttttttt aaataatcgt 15660atttgttttt attatggtta ggatggtttt tggggaatgg
taataagtag aattttttag 15720ggtgttggag ggatttatta ttttatttgt ttgttttgtt
attataataa aatattgtaa 15780ttgggtggtt ttgattaaag aaatttattt gtttatagtt
ttggagatta gaaatgtgag 15840gttaagttat tggggaaggt ttgttttttt ttgagttttt
tttttttggt ttgtagatgg 15900ttatgttttt gttgtttttt atatggtatg tatttttggt
attttttttt gtgtttaaat 15960tttttttttt gataaggata ttagttgtat tggattaggg
tttattttaa ttattttatt 16020ttatttaagt tattttttta aaggtcgtgt ttttattata
gttatatttt gggatattgg 16080gggttagggt ttaaattaat gaatttttag gggatgtaat
tgagtttata agataatttt 16140attttaaaaa gtttttttat ttttcgttat agtttgttat
agtttttaaa atttaatttg 16200agattttttt gtttatagat gagagtttat gtttttaatt
aggggatttt tgaagtttat 16260ttgagttggt tatagtggtt ttttttagta ttatttttta
ttttttttaa aattagtttt 16320attattttta atatagtttg aggttatttt tttgtttttt
agaagagttg tagtgttgtt 16380tggttgtttt tttatttatt aacggtgtta tatatttttg
tttttatttg tataagttaa 16440gtgtttatcg attttaagtt ttaggaaatc ggggtatatt
tattatgtat tgtagttttt 16500tggatttttt taaaatttta agtttgaaga attttttata
gtgtgaagtg ggaagttttg 16560ttttatattg agaggaatta ggatttgttg tttttatttg
tttattttta gggtataggt 16620ataaatttcg gttttataaa tatttttttt ttaaaaagtt
ttttttagag agggtttaag 16680gatattggta ttatgtagaa tttttttagg aatgatggtg
tggagggttt tggtttttag 16740ggtaagggtt ttttggagtt tagtgtgtag tgttagtgta
tgggggttgt gattgtgatt 16800ggtagtaagg tggttttgtt ggagttaagt agtagtgtgg
ttttagtgtt ttatttgaat 16860ttgtgttaag aaagtttgtg tttttggtcg ttagtagagg
tttgaatttt ttaataaatt 16920tttttttatt ttaaatgatt tagagtaaat tttattgttt
gtagttaaga attttgtgat 16980ttagatagtg attgtgattt atttttgttt gtgtgttttt
tagtgattag tagggtgttt 17040ggtgtaaagt agttgtttag aataggattt tatttgggtt
attttaggta tttttaagta 17100gaaagtattt tggtaagatt aatttatatt attgttgaaa
tgagtttatt tattgttttt 17160taggggttga gaaagggatg tttagagtag gtgtggttag
ttgtttatag ggaggaaggt 17220aaatgaaatg agtagtgttt ttcgggtgtg tgatagttag
ttttgtttaa atttgtttga 17280ttagttttta gtgttgattt tattttaatt gttattttta
tagatggaat taatttaatt 17340ttttttaatt atttattttt ttttttattt attttaattt
tttttaaaga tatagttttg 17400ttttgttgtt taggttggag tgtagtggta ttattatagt
ttattgttat ttttaatttt 17460tagatttaat tgattttttt atttaagttt tttaagtagg
tgggattata ggagcgtgtt 17520attatattta gttaattttt agattttttt tgtagagata
gggttttgtt atgttgttta 17580gtttggtttt gaatttttgg gtttaagtga tgtttttgtt
tttttttttt aaagtgttgg 17640gattataggt ataagttatt atgtttaggt tgattattat
tttaatggtt atattttatt 17700ttatggaatg atatggatta ttatttattt ggttaggtat
tttttgttga atatttaggt 17760tgtttttgat tttttttttt ataattagaa atattgggtt
gggtatggtg gtttatattt 17820gtaattttag tattttggga ggtcgagacg ggcggattac
gaggttagga gatcgagatt 17880attttggtta atatggtgaa atttcgtttt tattaaaaat
ataaaaaaat tagtcgggcg 17940ttatggcggg cgtttgtagt tttagttatt cggaaggttg
acgtaggaga atggcgtgaa 18000ttcggaaggc ggagtttgta gtgagcggag atcgcgttat
tgtattttag tttgggcgag 18060agagcgagat tttgttttaa aaaaaaaaaa aagaaacgtt
gttgaaattt aggtttttta 18120gataaaaatt ttttatattt atattaattt ttttaagata
aatttataga ggtcgggtga 18180ggtggttcgt atttgtaatt ttagtatttt ggaaggacga
ggtgggtgga ttacgtgagg 18240ttacgagttc gagattagtt tgattaatat ggagaaattt
cgtttttatt aaaaatataa 18300aattagtcgg gcgtggtgga gtatgtttgt aattttagtt
atttcggagg gtgaggtagg 18360agaattattt gaaatcggga gacgaaggtt gttgtgagtt
gagattacgt tattgtattt 18420taatttgggt aataagagcg aaattttatt ttaaaaaaaa
aaaaaattat aaaggtagaa 18480tttttgggta aaagaaaatt ataatttttt tagattatta
tttgttatta tgatatgtgg 18540gaaatgtttt gttttaattt attttttttt gttaattggt
aagattagat tttttttttt 18600tttttttttt ttttttttga gacggagttt cgttttgttt
tttaggttgt agtgtaatgg 18660tgtaatttta gtttagtata agttttattt tttaggttta
cgtttttttt ttgttttagt 18720ttttcgagta gttggaatta taggtatttt ttattatatt
cggttaattt tttgtatttt 18780taatagagac ggggttttat tatgttagtt aggatggttt
tgattttttg atttcgtgat 18840ttgttttttt cgggttttta aagtgttggg attataggcg
tgagttatcg tgtatagttt 18900agattttttt ttgtatatgt attgattaat tgtttttttt
tttttgtgag tattttgttt 18960atttatgttt tttagttatt tttttgtttg ggttatttat
attttttttt tggattagtt 19020aaggttttta atattaaaga tattaatttt taagtgttat
atatgttaaa atattttttt 19080gatttttttt atggttagta tatttatttt ggagttagaa
ttaaggaatg agaaattttt 19140tttatttata tttttatttt ttaaatgata tgttttgata
tgtagagatt ttaaatttta 19200attgattata gttatattat ttttatttgt gggttagtat
tataagtttt aaagaatatt 19260ttaagattta tggagttata tagtttttgg gttgatgttg
tggtaggagt tgttatttat 19320ttttagggtt ttttgttttg ttttgttttt tgtttttttt
tgagatagag ttttgttttg 19380tcgtttagga tggagtgtag tgttgtaatt ttagtttatt
gtaattttta tttttcgggt 19440ataagcgatt tttgtgtttt agttttttta gtagttgggg
ttataggtat gtattattat 19500gtttggttaa tttttgtatt tttagtagag atagggtttt
attattgttt aggttggttt 19560tgaacgtttg attttaagtg atttgtttgt tttggttttt
taaagtgttg ggattataag 19620tatgggttat cgtgttttgt tattattttt agtgttaaaa
gaacgttaga taaattaaat 19680ttaatagagt ttgattgagt aaagaattat ttgtgtgaat
taggtagttt ttaggtaaaa 19740ataagtttag agagattttt gtattgtttt atagttaaag
atttatggat agaaaaagga 19800aagtgatgta tagaaattgg aagtgaggta tagaaataga
cggattggtg atggtatggt 19860gtttggttta tttgagttta gttttaatag ttggtcgttt
gtgagtggtt gaagtttggt 19920tgttgtgatt aattgagatt cggttatttg gtacgagagt
aggtgatagt ttgtttatac 19980gtgtagttag tttatagttt attatgtata gagaaattat
tagggtgaat ttaaaatatg 20040taaggagaag gttttaggtt aaatttgatt taatattagg
atcggtgggg ttttttttta 20100ttagttttta agttgatgat ttatttttta tttatttttt
tttttatcgt gttttagagt 20160ttttttaaat tatgattttt ttttttaaat tttttattta
attgtttatt ttatacgtag 20220aatataattt tatttcgtat tttatagaga ttagaggtga
aaattgtatt tttattttta 20280ttttgtatat tgttttttat cggtttttgt tttttttatt
ttttttttgg gttttttttt 20340tattttaaat acgtaatacg gattttattt ttttttttat
cggtttttat ttttttttag 20400ggattttttt ataaaagtta gtttttttaa gataagtagg
tatgaaggga tagatggaaa 20460agtgtttagt gttcgaggat tgtttaattt ttagtttggt
ataagttagt ttttttgagg 20520gtttatttat attttggtaa ttttattttg tagagtttaa
tttttgtgat attttgtggt 20580aggattcgtt ttggtattcg ttaggatttt gttttgttgg
aaatcgaatt aaaatgagta 20640tgggtaaaaa gagacgtttt tagtttaagt gttaagttta
gaggtggatt agtttttgga 20700ttgcggggtt tagtttagga tagatattat gagagttttt
attttgtaga aattagaaag 20760atagattttg tttttattta tggatttatt tttgtataat
tttttgtttt aaaaatataa 20820taagataaat tttttatgtt aaaggatgta gtagagggtt
tttttatagt aatagaaatg 20880ttgtaggatg aattttttat atggagtgag ggtgacggtg
ggatagagat tttggatatt 20940taggtaaaga attgggttgg aaaaggaagt tattaataga
atagcgagaa agaatattgt 21000aaatgttgtg agttatagtt attttggata tagtgttata
agagtgtatt ggaatgtatt 21060gaggttgata agttagggat taggaagatt gttggtgaat
aatttttttg gttttttgta 21120aggaatttgg aaagtttatt tagagaggtg tgaaaatagt
agaaaaaata ttgggataaa 21180gaaacggggt aaaagtaaat atgttttaga ttagtcgttg
agaaatttaa gaaataaaat 21240taggttttgt tttagagaaa agggagattt gggtattttt
ttggaggttc ggaagttttt 21300tgtttgttaa gtggattaaa gggtatttgg ttaaggattg
tgtgtttata gtttaaagta 21360attatgtata gaatttagga attatttgtt ttttacgttt
ttaatggatt gttttagaga 21420ttgttttttt ttgtgaagtt gatgtttgtt ttgtgttgaa
tgtaatatag ttataaggag 21480gttatcgtga tagggagttt gtattttttt tttggatgtg
gtatttaaag tgttgaataa 21540ttagtatggt ttaattattg attttttagt ttagttgtta
gaagttagtt tgggggattt 21600tattgtgcga ggttttattt tttttgtttt tggatgatgt
tgaggtttgg aggggttagg 21660atttaatatg ttgagtgggg gtatttagga gtgttgggta
gtagttacgt atttattagt 21720atatttgaat tgatttatat tagtattata ggtttttatt
ttttttatag agtatatgtt 21780aagtaaatgt agttattgaa ttagatagaa gaacgttagt
tagtaatttt ttgtttttat 21840tatgagtagt gttttttaga attattttag ggtggagggt
tgagaggtgg gaagggagtt 21900ggagtttttt atgtttatat atgtattatt ttagggattt
tttagtaata taataaaata 21960ggtttttttt cgtttatttt attagtgggg tattgaggtt
ttaggatgga aataattatt 22020tagggttttt agatggtgag aaagagtgag aatttaaatt
taggtttgag atgaagtagg 22080gatttgtgtt attatttcga acgttattat taagtatgga
aataaaggaa aattttgagt 22140gtttttaagg gaaattttag gtatttagtt agttttgaga
agtaagtaag taatttgtta 22200ggtaggaagg taatagtagt ttaaaataat agttaaggaa
gttagagttc ggagatgttt 22260agttttagga attaaagata atattttaat atacgttttt
gagtttgtta tttagaaatt 22320tggattttta tgtggatttt tattaaacgg atttgttggt
ttgtagattt tagatagagg 22380gagttgagga ttgtattttg attatttttt tttattttaa
atttttttcg gaggtttttg 22440aaagttatat ttatgagtta aagttaatat ttttttttgt
taattttgaa tttttaggta 22500aggtatcgtt ttttgaatta attataaatt aaagaatttt
tgaatttatt tatgatttgt 22560aagtttttgt tttaagatat tttatttttt ttaggttaaa
ttaatgtgta ttttttatgt 22620attgatttat aattttattt aatttttgtt gtttttttga
aaatttattt cgtttttaaa 22680aatttttatt tgtgtgaaag gaaattatgt tggttttttt
aaattattaa gttaaagaga 22740aaatttaagt tgggaattgt ttagggtaaa tttgtttcgt
attttattta aagttatttt 22800tttgtttatt gagataaatg tatatttgat tgtttttttt
ggaaaggtta attagaaatt 22860taaaagaatg taatcgtttg ttttttattt atttgtgatt
tggaagttta ttgtttgttt 22920taagttgttt tgttttttta gattaaatta atgtttattt
tatatatgtt gattgatggt 22980ttatgttttt ttaaaaggta taaaattaag ttgtgttttg
attattttgg gtatatgtcg 23040ttagaatttt ttgagggtgt gttacggggg tatgttttta
attttggtaa aataaatttt 23100ttaaattgat atttgtttta gatttttagg gtttatattt
gttagttatt agagagttta 23160ggttttgagt atgcgttgtt tggttttttt tgtttagggt
tttgtgaata aatatttttt 23220tttttagtgt tgtaatttta gtgtggatat ttggatttaa
ttgtattggg taagtcgatt 23280ttaatttgtt aagtacgttt atgtgaagaa attattaaat
aggttttgta tgaggaataa 23340aatttttaat ttatttgggt gtaggtgggt tgagttcgaa
aagagttagt aaagggagat 23400ggggaaggga ttgttttata ggagttgggt aggtaatgga
aagttatagt aaaacgtggt 23460tatttattgt tagtagagga gggggttata aggtatatag
tggggagatt ataagattta 23520ttgtttagaa gaagaatgtt ataaagttgg ttgattagtt
aaggtagggt agggataagt 23580tataatggta aaatgttgta attttggtta tttagttaag
gtaggaattg gttgttttat 23640tttgtgggtt ttttggttgt tttagatttt ttggtttttg
taggttattt ggatatatat 23700gtgtaggtta taggggttat aatagttgag ttttagttta
gaggtttgat ataatttagt 23760tatataatag ttttaatgat ttaaaagtta gtattttttt
tttttttttt tttttttttt 23820ttttgagatg gagtttcgcg ttgtcgttcg ggttggagtg
tagtggcgtt attttagttt 23880attgtaattt tcgttttttg ggtttaagtg atttttttgt
tttagttttt tgcgtagttg 23940ggattatagg tatatgttat tatgtttagt taatttttgt
attttttttt tagtagagat 24000ggggttttat tatgttggtt aggttggtta taaatttttg
attttaaatg atttgttatt 24060tcggtttttt aaagtgttgg gattataggc gtgagttatt
acgtttagtt aagtttttgt 24120tttttttata gttagggttg ttttttttat atatttgttt
aaagggttgg tagtatttgt 24180atttaagtga agagatttaa gtatttttga gttttagagt
agtaggtggg ggttattatt 24240agtatggggt tttttatgcg ggagatttta tagttttttt
tttttttttt tttttttttt 24300tgataatgtg tttaggtttt ttatagtttt tggtattaag
aaattttaat agatgttaat 24360tagtttggta atttagtata gcgttagtat taagtcgtta
aggttgagga tatgttatcg 24420tgatatattt ttaggaggtt ttgataatat gtgtttaagg
tagttagagt atagtttggt 24480tttatatttt ttagggagat atgagttatt atttaatata
tgtaaaatga atattggttt 24540agtttggaaa ggtaggataa tgtgaagtag gtagggggtt
tttagattat atatgtgaga 24600gataaacggt gttattttta aatattttta aattattttt
ttttttagtt atttattagg 24660aggaaagtaa atgtttgtta gatatggtta ttaattatcg
tcggtgggtg ggatgaaaag 24720gagtttattg atgattggtt aggtgtttat ttttattagg
ttttagattt ttttttggtt 24780aatgagggag ttaatttttt attgggggta gtaagtttaa
atgttcgggg taagttaggt 24840aatataattt agtgaagtag gtatgatgtt tgtatatgaa
gtatttatga gttttatttt 24900tttaagtaaa taggtttttt tttatttttt ttgaaatgta
tattattgtt agtttgtttt 24960tttttatttt aattttgata gagatatatg tataaaaaat
atatttttag gttaggagta 25020gtggtttata tttataattt tagaattttg ggaggtttag
gtaggaggat tgtttgaggt 25080tagaaattta agagtagttt ggataatata gtgagaattt
tatttttata aaataaaaaa 25140attagttaaa ttggaggtaa aggcgggtgg atggtttgag
gttaggagtt taaaattagt 25200ttgattaata tggtgaaatt ttattattat taaaaatata
aaattaggta gggtatggtg 25260gtttataatt gtaattttag tacgttggga ggtcgagacg
ggtggattat gaggttagga 25320gatcgagatt attttggtta acgtggtgaa atttcgtttt
tattaaaaat ataaaaaaag 25380ttaggtgtgg tcgcgggtgt ttgtagtttt agttatttag
gaggttgaag taggagaatg 25440gcgtgaattc gggaggcgga gtttgtagtg agttaagatt
acgttattgt attttcgttt 25500gggtgataga gtaagatttt gttttaaaaa aaaaaaaaaa
aaaaaaatta gttaggtttg 25560gtggtgtata tttgtaattt tagttatttg ggaggtagga
gtattatttg aatttgggag 25620gtggaggttg taatgggtcg agattatgtt attgtatttt
cgtttgggta ataagagtaa 25680aattttattt taaaaagata ggataggata cgatacgata
taataagacg agaagaaata 25740ttatgttttt tataaaaatt ttttagaaaa taaaaaagga
tagattatgt tttaattttt 25800ttaatgaggt tagtgttatt ttaatattaa agttaaataa
atatattaaa ataaataaaa 25860attatagaat aatatttttt atgaatagag atgtaaatat
atttaataag atattagtaa 25920ataaaaattt attaatatgt ataaaagatt atatattatg
attaagtatg atttatttta 25980gaaatgtaag gttggtgtga tatttaaaaa ttaagtaatg
ttttttttag tatatggtga 26040ggtttttaaa attaattaat taaaaataat aaaattttaa
aattagtgaa tgtaatgtat 26100tagattaata gattaaaata tattatattg ttattttaat
agatatagaa gtatttgata 26160atagttaata tatattcgtg gtaaaaattt ttaataaatt
aggaataaaa gtaaattttt 26220ttaatttgat agagggtaat tatagataat ttatagttaa
tgtatttaat gatgaaagat 26280agaatgtttt ttatttaaga ttaggaaata ggtaagaatg
tttattttta ttatttttat 26340ttaatattgt attattggtt atagttaggg taatttaaaa
agggggtaga gggagaaaga 26400gagagaaagg taaattaaag atatttagaa tgaaaagaaa
aaagtaaaat tgtgtttatt 26460tgtagattat atgatttgta tgtagaaaat tttaagtaat
ttattaaaaa attttattgg 26520aattaataaa tgagtttagt aaataaggtt taagtatata
aggtcgatat gtaaaaatta 26580attgtatttt tatatattag taataaataa tgtgaaaatg
aaattaagga aataaaattt 26640tatttatagt aatatttaaa agaataaaat atttagaaat
gagtttaatg aaagaaatgt 26700aagatttata ataaaaatta taaaattttg tttagagaaa
ttgaagaagt tttaaattag 26760aggtagtaaa ttatagttta tggttttgaa ttggtttttt
gtttgttttg taaataaagt 26820tttattgaaa tataattttt ttttttttga gatggagttt
tgtttttgtt gtttaagttg 26880gagtgtaatg gtgtgatttt agtttattgt aatatttatt
ttttaggttt aagcgatttt 26940tttgttttag tttttcgaat agttggaatt ataggcgtgc
gttattatgt ttggttattt 27000tttttgtatt tttagtagta acggggtttt tttatgttag
ttagttggtt ttgaattttt 27060gattttaggt agtttattcg ttttagtttt ttaaagtgtt
gggattataa gtatgagtta 27120tcgcgtcggg ttaatattgt ttttatatta taaagtaaag
ttgaatatag tttataatat 27180tataaatatt tttattattt ggttttttgt agaaaatttg
ttaatttttg atttaaataa 27240ttttatgttt atagattaaa agattttatt gttaagatga
tagttttttt ttaatttatt 27300gataaatttt tgttaattta aaaaaaatta atgtcgtttt
taagaaagtg agtttatttt 27360aaatattgcg aaattaaggg aaaaaattaa aaagttagtt
ttggttgggt gtagtggttt 27420acgtttgtaa ttttagtatt tgggaggtcg aggtaggtgg
attatttgag tttaggtgtt 27480taagattagt ttggttaata tggtgaaata gtgattttat
taaaaatata aaaattattt 27540gggtatggtg gtatttgttt gtaattttag ttattaggga
ggttgaggta ggagaatcgt 27600ttgaatttgg gaggcggagg ttgtagtgag ttaagattgt
gttattatat tttagtttgg 27660atgatagagt gagatttggt tttaaaaata aaaataaaaa
aaatagttta gtaggttttt 27720gtggaaattg ataagttttt gttaaaattt atatagaaaa
gcgaatgatt taggatagtt 27780taaatatttt gaaaaagaat aaagttaaaa aaaaaattta
tattatttaa ttttaatatt 27840tgttattgtt aaagaaaaat tttgtattag atatttgtta
aaaatgttaa gatagatttt 27900atttagatta ttgtggtagg gaagagatat tttagtatta
attgagttta attttattaa 27960attagaaggt aagaggtttt ttaaatgtta ggatgtgtta
agggaaaagt attgaaggat 28020gttagttgag gggagggagg tttggttaat gagtatattg
attagattgt gaatttgtgt 28080ttattaaatt gtgaattggt gtttattaat attagggttt
tattttatta ttaagattgg 28140gagatagagg ttttgtttta tttaatgatt atatttcgaa
aggatggttt ttaggttttt 28200gggaaagata tttttgtgtt atagaagata tatttttaag
agatagagaa aggattttta 28260attgttagtt ttttaaagta aattttttag gaaaaggggg
ttcgggggtt tatatttagg 28320ttttgtttgg aataaattat aaattttttg ggtagtattg
aattttatta ggtagaaatt 28380taagggggtt gaagttatta ttttaaggat gtggttatga
attgttagaa ataatagtgt 28440ttgttgaagt tttttagtgt tagaggtgga tgaaattatt
tgtgttgaga gtttgtagtt 28500tgttttttta tttatatttt attttttatt atttattttt
tttttttttt ttttgagata 28560gggttttatt tttgttgttt aggttggatt taggttggag
tgtagtggcg ttatttcggt 28620ttattgtaag ttttgttttt cgggtttacg ttattttttt
gttttagttt tttgagtagt 28680tgggattata ggcgtttgtt attacgttcg gttaattttt
tttgtatttt tagtagagac 28740ggggttttat tacgttaatt aggatggttt cgattttttg
attttatgat ttatttattt 28800cggtttttta aagtgttggg attataggta tgagttattg
cgttcggttt attttttttt 28860tttttttaag tataatatat ttttattaag ttgaatagaa
atattaataa agattagtat 28920gtatagtgat tatagatgta gatagataaa aaagaaaaag
ggatataagg gcgtattgaa 28980tttatgataa tattgtttta gtattatagt aggagttaat
ttattttagg gttggttttt 29040taagattagt ttcgggaagt ttgatttgtt tagttttgat
tattggttga tcgggattgg 29100ggatttagtt ggtaggatag gttgtagttt ttatttttat
tgggttgttt tttgatttag 29160aattaggttt ttagtttttt tttaggatga aggttgtaat
gatttgtagt tatttgagta 29220gttgggtttt tttttttggg agtttattat atttttattt
atattattat ttatcgtagt 29280gtttagtatt attaggtcgg tgaggaatgt gttaaggata
gggataattt tttttttttg 29340ttgttgttat tttgtatggg ttttgtgggg gtttgttttt
agggtggtcg atttggaggt 29400ttttatattg atgagtagat ttttgttcgg tggtttgttt
ttgttgtata gttttggata 29460tttttttgtt gttttatttt ggttatttag agtagagtag
ttataatttt ttattttata 29520gtgtttgggt taaagtggtt taggttaggg tttttgaaag
acgagatatt agagagtttt 29580tttttttttt tttttttttt gttttttata tttttttttt
aggagtataa agaggttatg 29640tgagtacgta gtgggagggt ggttatatgt gagttaagag
aggggtttta gaatgaagtt 29700tgtttttttt gtattttgat cgtggatttt tagtttttag
aattgtgaga aatgaatttt 29760tgttatttaa gttttttagt ttgtggtatt ttgttatggt
agtttgagtt aattaatata 29820ataggaaaga aatattttaa ttttcgtaaa tttattttta
agtttattaa tgtttaaagt 29880gattaatagt gggtatattt tgtaaattat taagatttta
tatagttata ttttatgatt 29940ttttataatt agttggttat ggtttgtaat ttttgataaa
tattgttttt agttttagag 30000921DNAArtificial SequenceChemically
Synthesized - primer 9gtttaggttg gagtgtagtg g
211025DNAArtificial SequenceChemically Synthesized -
primer 10atcctaacta acataataaa acccc
251127DNAArtificial SequenceChemically Synthesized - primer
11agttttttta tttaggggtg gatttta
271227DNAArtificial SequenceChemically Synthesized - primer 12ccaaactaat
ctcaaatacc taacctc
271325DNAArtificial SequenceChemically Synthesized - primer 13gttatttagg
cgggagtgta gtaac
251425DNAArtificial SequenceChemically Synthesized - primer 14taaatcataa
aatcaaaaaa tcgaa
251525DNAArtificial SequenceChemically Synthesized - primer 15tatttaggtg
ggagtgtagt aatgt
251625DNAArtificial SequenceChemically Synthesized - primer 16taaatcataa
aatcaaaaaa tcaaa
251724DNAArtificial SequenceChemically Synthesized - primer 17aaatcataaa
atcaaaaaat cgaa
241824DNAArtificial SequenceChemically Synthesized - primer 18aaatcataaa
atcaaaaaat caaa
241924DNAArtificial SequenceChemically Synthesized - primer 19ttatttaggc
gggagtgtag taac
242026DNAArtificial SequenceChemically Synthesized - primer 20ataaatcata
aaatcaaaaa atcgaa
262125DNAArtificial SequenceChemically Synthesized - primer 21tttttgagta
gttgggatta taggc
252223DNAArtificial SequenceChemically Synthesized - primer 22acactttaaa
aaaccgaaac gaa
232326DNAArtificial SequenceChemically Synthesized - primer 23ttttttgagt
agttgggatt ataggt
262425DNAArtificial SequenceChemically Synthesized - primer 24caacacttta
aaaaaccaaa acaaa
252524DNAArtificial SequenceChemically Synthesized - primer 25aacactttaa
aaaaccaaaa caaa
242623DNAArtificial SequenceChemically Synthesized - primer 26aacactttaa
aaaaccgaaa cga
232727DNAArtificial SequenceChemically Synthesized - primer 27gttttttgag
tagttgggat tataggt
272824DNAArtificial SequenceChemically Synthesized - primer 28gagtagttgg
gattataggc gttc
242925DNAArtificial SequenceChemically Synthesized - primer 29taaatcataa
aatcaaaaaa tcgaa
253025DNAArtificial SequenceChemically Synthesized - primer 30agtagttggg
attataggtg tttgt
253125DNAArtificial SequenceChemically Synthesized - primer 31taaatcataa
aatcaaaaaa tcaaa
253225DNAArtificial SequenceChemically Synthesized - primer 32ttgtaatttt
agtattttgg gaggt
253325DNAArtificial SequenceChemically Synthesized - primer 33ctcttattac
ccaaactaaa cccaa
253424DNAArtificial SequenceChemically Synthesized - primer 34tgttgtttag
gttggagtgt aatg
243526DNAArtificial SequenceChemically Synthesized - primer 35ctataatccc
aacactttaa aaaaac
263625DNAArtificial SequenceChemically Synthesized - primer 36tttgttgttt
aggttggagt gtaat
253727DNAArtificial SequenceChemically Synthesized - primer 37cacctataat
cccaacactt taaaaaa
273824DNAArtificial SequenceChemically Synthesized - primer 38gtaagtttcg
tttttcgggt ttac
243926DNAArtificial SequenceChemically Synthesized - primer 39cctaactaac
ataataaaac cccgtc
264025DNAArtificial SequenceChemically Synthesized - primer 40taagttttgt
tttttgggtt tatgt
254125DNAArtificial SequenceChemically Synthesized - primer 41ctaactaaca
taataaaacc ccatc
254224DNAArtificial SequenceChemically Synthesized - primer 42tttcgagtag
ttgggattat aggc
244323DNAArtificial SequenceChemically Synthesized - primer 43aacactttaa
aaaaccgaaa cga
234426DNAprimer 44ttttttgagt agttgggatt ataggt
264525DNAArtificial SequenceChemically Synthesized - primer
45caacacttta aaaaaccaaa acaaa
254625DNAArtificial SequenceChemically Synthesized - primer 46ttttttgagt
agttgggatt atagg
254730DNAArtificial SequenceChemically Synthesized - primer 47aaaacaaact
aacaatatcc ttaaaaatta
304825DNAArtificial SequenceChemically Synthesized - primer 48attgtgggtt
agttataatg gttta
254923DNAArtificial SequenceChemically Synthesized - primer 49actcactaca
aactctacct ccc
235025DNAArtificial SequenceChemically Synthesized - primer 50gtgggttagt
tataatggtt tacgt
255122DNAArtificial SequenceChemically Synthesized - primer 51actacaaact
ctacctcccg aa
225225DNAArtificial SequenceChemically Synthesized - primer 52gtgggttagt
tataatggtt tatgt
255324DNAArtificial SequenceChemically Synthesized - primer 53ctcactacaa
actctacctc ccaa
245426DNAArtificial SequenceChemically Synthesized - primer 54tgtagtggtg
tgatttttgt ttattg
265525DNAArtificial SequenceChemically Synthesized - primer 55tataatccca
acaccttaaa aaacc
255624DNAArtificial SequenceChemically Synthesized - primer 56gtttaggatg
gagtgtagtg gtgt
245725DNAArtificial SequenceChemically Synthesized - primer 57tgggattata
ggcgtttgta attac
255822DNAArtificial SequenceChemically Synthesized - primer 58acaccttaaa
aaaccgaaac ga
225927DNAArtificial SequenceChemically Synthesized - primer 59tgggattata
ggtgtttgta attatgt
276023DNAArtificial SequenceChemically Synthesized - primer 60acaccttaaa
aaaccaaaac aaa
236125DNAArtificial SequenceChemically Synthesized - primer 61tgtaagtttc
gttttttggg tttac
256221DNAArtificial SequenceChemically Synthesized - primer 62aaccgaacgc
tatatctcac g
216325DNAArtificial SequenceChemically Synthesized - primer 63taagttttgt
tttttgggtt tatgt
256424DNAArtificial SequenceChemically Synthesized - primer 64aaaccaaaca
ctatatctca cacc
246526DNAArtificial SequenceChemically Synthesized - primer 65tagggttatt
tgggttatag atttag
266625DNAArtificial SequenceChemically Synthesized - primer 66cctaactaaa
tcacatttac cattc
256730DNAArtificial SequenceChemically Synthesized - primer 67attatagtaa
taggtgataa tttaagagaa
306829DNAArtificial SequenceChemically Synthesized - primer 68aaatacctct
tattaaacac tcaaattat
296929DNAArtificial SequenceChemically Synthesized - primer 69gagaaattat
gtttggttag atttttaat
297029DNAArtificial SequenceChemically Synthesized - primer 70ataaccaacc
caatactact ttaatatac
297125RNAArtificial SequenceChemically Synthesized - PPP1R8 gene
71ggaacucgaa ccuccacgaa caauu
257221RNAArtificial SequenceChemically Synthesized - EZH2 siRNA
72aagacucuga augcaguugc u
217321RNAArtificial SequenceChemically Synthesized 73aacuggacuu
ccagaagaac a
217421DNAArtificial SequenceChemically Synthesized - primer sense
74gtttaggttg gagtgtagtg g
217525DNAArtificial SequenceChemically Synthesized - primer antisense
75atcctaacta acataataaa acccc
257627DNAArtificial SequenceChemically Synthesized - primer sense
76agttttttta tttaggggtg gatttta
277727DNAArtificial SequenceChemically Synthesized - primer antisense
77ccaaactaat ctcaaatacc taacctc
27
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